From analia_alet from intech.gov.ar Sun Mar 2 05:17:34 2008 From: analia_alet from intech.gov.ar (Analia Alet) Date: Sun Mar 2 16:29:22 2008 Subject: Protein unavailable for IMAC binding In-Reply-To: <200803011703.m21H3wL19701@net.bio.net> References: <200803011703.m21H3wL19701@net.bio.net> Message-ID: <1204453054.47ca7ebe96ff2@webmail.intech.gov.ar> Once I had a problem like this with BL21(DE3) trying to express a protein with a 6xHis Tag. And I tried induction with lactose to allow protein to folder properly. It takes much more time, but it could work. Another thing to do could be to make the culture in M9 supplemented with casein-aminoacids (CAA)1% P/V so you have under control the "food" the strain is "eating" (i don?t remeber the properly words in english) Hope it works Best regards Anal?a DK wrote: > In article <47c736ea$1@clarion.carno.net.au>, Bean Long wrote: >> Hi all, >> >> I'm expressing several proteins in BL21(DE3) using 0.5 mM IPTG for >> induction. One protein is proving a little difficult in that it is >> successfully extracted into the soluble fraction but only a relatively >> small proportion of this is subsequently bound to IMAC. > >> I say relatively >> small in that a huge amount does not bind to IMAC but recovery of pure >> protein is still quite good. It would seem that a large amount of the >> protein is forming soluble, yet unbindable (occluded tag), aggregates. > > Absolutely correct. This kind of things happens all the time with > MBP fusions (MBP is so soluble that it keeps any garbage in > solution and from forming inclusion bodies). > >True, but I think the OP meant His-tag. >> I wondered if using lower IPTG concentrations would reduce the total >> protein concentration and therefore the likelihood of aggregation or is >> there another approach? >Yes, it may. Although it is not the reduction of total protein >concentration that helps, it is the rate of protein synthesis - the >protein needs time to fold properly, and partially folded protein tends partially folded protein present at any one time, hence less likely to > Before that, characterize fraction that does elute. In numerous cases > here, the bound/eluted fraction was still an aggregate that precipitated > immediately after MBP cleavage with rTEV. > >> I do not wish to use denaturing agents (e.g. >> urea or guanidine) during purification as the protein is bound for >> crystallisation. > > You could still try 0.5-1.0 urea, which is not denaturing for a vast > majority of proteins but sometimes disrupts aggregates very efficiently. > > DK From ben.long from yourfinger.anu.edu.au Sun Mar 2 17:24:15 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Mon Mar 3 13:29:46 2008 Subject: Protein unavailable for IMAC binding In-Reply-To: References: <200803011703.m21H3wL19701@net.bio.net> Message-ID: <47cb290d$1@clarion.carno.net.au> Many thanks to all for your posts. I've got a few ideas to play with now. We have tried low urea concentrations with this protein before, so that may be an initial and simple approach. Cheers, Bean From hutsugi from fhcrc.org Sun Mar 2 16:40:01 2008 From: hutsugi from fhcrc.org (Utsugi, Heidi K) Date: Mon Mar 3 13:29:52 2008 Subject: Methods Digest, Vol 34, Issue 2 References: <200803021704.m22H4ML14570@net.bio.net> Message-ID: I will remain! There is always something to learn from ones peers and humility has always been evident here. Martin Luther King, Jr. once said "In the end, we will remember not the words of our enemies, but the silence of our friends." Heidi ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu Sent: Sun 3/2/2008 9:04 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 34, Issue 2 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-request@net.bio.net You can reach the person managing the list at methods-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. Re: PCR Problem Debate/Foolishness (DK) ---------------------------------------------------------------------- Message: 1 Date: Sat, 01 Mar 2008 05:51:22 GMT From: dk@no.email.thankstospam.net (DK) Subject: Re: PCR Problem Debate/Foolishness To: methods@net.bio.net Message-ID: In article <1204332997.7459.0@proxy02.news.clara.net>, ChenHA wrote: >Jose de las Heras wrote: > >> >> wtf? >> >> I never thought I'd have to use the killfile on this group... but here it >> is. Have fun. >> > >Thank you. Sore that I pointed out to you that you can't tell nonsense >assertion in science from real science? The mediocre will always stick >together. > >Don't worry, everyone else. I don't frequent this newsgroup much >anymore, so you can go back to you gentle waffling of nonsense. There >are just a couple of individuals who have always been good scientists >here, the intelligent ones will recognise who they are and I hope I had >help you recognise who the idiots are (no, not me, I may be obnoxious, >but I sure ain't no idiot). > >Last post here. Do sigh a relief. That's too bad. You were knowledgable and valuable contributor in the past. It's sad to see a real life persona we knew taken over by a drama queen. Than again, to each its own. Good luck. DK ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 34, Issue 2 ************************************** From aawara from pontiff-playground.org Sun Mar 2 21:03:36 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Mar 3 13:29:57 2008 Subject: Question on recombination in E.coli and in baculovirus References: <%xGyj.88$8m2.34@newsfe05.lga> Message-ID: In <%xGyj.88$8m2.34@newsfe05.lga>, DK wrote: > I am considering co-expressing three proteins from the same > baculovirus. For that, a transfer plasmid from Novagen is > available which has two polh promoters and two p10 promoters. > Each of the same promoters is found on different strand > (i.e., one strand has one polh and one p10 and another strand > has one polh and one p10) "to minimize recombination" according > to the company. Promoters are ~ 70 and 90 bp. > > More than that, I might need to express two of the proteins > as fusions with the same small protein (162 bp). There is no > problem placing those on different strand. > > Because I don't really know much about homologous > recombination in either system, I worry if there are caveats > related to recombination and resulting instability of the > 1) transfer vector in E.coli 2) resulting baculoviral DNA > in the insect cells. I wouldn't worry about insect cells - given the time length of expression, after infection with baculovirus (or you concerned about recombination when you make the virus?). In E. coli, if I understand you correctly, the repeats will be inverted (the polh and p10 promoters are on opposite strands, so they must be inverted with respect to each other, correct?) I don't think you'll have major issues with stability, but if you do, propagate the plasmid in an E. coli strain like STBL2. We have had very good luck propagating retroviral vectors, and retrovirus clones in this strain, and both of these types of clones have two copies of direct repeat, the LTR (U3RU5), and also four copies of inverted repeats - at the U3 and U5 ends. The direct repeats LTRs range in size from about 300 bp to 1 kb, and the inverted repeats are 15 - 30 bp in length. Anyway, these constructions are completely stable in STBL2. I think we originally obtained this from Invitrogen. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From jennyf from nowhere.com Mon Mar 3 01:00:05 2008 From: jennyf from nowhere.com (Jenny F.) Date: Mon Mar 3 13:30:01 2008 Subject: Source for Wizard/Merlin midiprep plastic columns? Message-ID: Hello everyone, We use these columns to do a homemade Merlin/Wizard-type midiprep. (In fact we just reuse the columns after getting rid of the resin.) Unfortunately, we've lost our last of these columns. Promega has stopped selling the type that we use -- they've seemingly redesigned the columns so as to prevent reuse. (They now have the resin collected in a narrowed part of the column toward the bottom, which you are supposed to physically separate in order to later elute... clever devils! Unfortunately, these new columns also work extremely poorly.) The type that we use is perhaps 50 mL total volume, bottom maybe 4mm diameter, bottom opening maybe 2mm diameter (this is important so they fit into the vacuum manifold). They also have a relatively thick plastic frit at the bottom (important so that resin can't get through), which is the full size of the column diameter. Anyone know of a source for these columns? Thanks, Jenny From m.davies from herts.ac.uk Mon Mar 3 05:00:35 2008 From: m.davies from herts.ac.uk (Matthew Davies) Date: Mon Mar 3 13:30:06 2008 Subject: Surfactant like coating for glass (to stop PS beads binding) Message-ID: I'm a microfluidics researcher trying to develop a bead based separation method. I have a problem with non-specific binding of biotinylated beads over a glass slide (forming the base of the channels) partially coated with streptavidin. Plain glass (slide cleaned with 1:1 MeOH/HCl for 30 minutes). I have tried the manufacturers suggestion of running the beads with blocker & surfactant to prevent binding and it worked fine. I have determined that its the surfactant that prevents the binding. Priming the channel with a surfactant solution doesn't help, but in the final application I cannot have surfactant in the solution. My hope is that there is a coating that I can apply, either by running a solution down the channel or by dip-coating the slide, that is compatible with streptavidin. I have to precoat the slide with streptavidin over a specific region, so any other coating has to come after. I know this is sort of a weird question for this forum. So thanks in advance for all the help. Dr Matt Davies Microfluidics and Microengineering Group STRI University of Hertfordshire PS resending this as it hasn't been posted yet and thought maybe it was because the first one wasn't in plain text format. From r06ma6 from abdn.ac.uk Mon Mar 3 07:13:16 2008 From: r06ma6 from abdn.ac.uk (Arianmanesh, Mitra) Date: Mon Mar 3 13:30:11 2008 Subject: Albumin depletion from sheep plasma Message-ID: Dears I need to deplete Albumin from sheep plasma while there is no defined and perfect method for it. Actually, I used several methods for that such as Immunoprecipitation and SwellGel Blue Albumin Removal Kit but I could not get good results. So, now I need to know that how to deplete Abumin from the sheep plasma. Can anybody give me an advise to do that well? Kind regards, Mitra The University of Aberdeen is a charity registered in Scotland, No SC013683. From k.schink from gmail.com Mon Mar 3 07:54:45 2008 From: k.schink from gmail.com (Kay Schink) Date: Mon Mar 3 13:30:15 2008 Subject: Question on recombination in E.coli and in baculovirus In-Reply-To: <%xGyj.88$8m2.34@newsfe05.lga> References: <%xGyj.88$8m2.34@newsfe05.lga> Message-ID: DK wrote: > I am considering co-expressing three proteins from the same > baculovirus. For that, a transfer plasmid from Novagen is > available which has two polh promoters and two p10 promoters. > Each of the same promoters is found on different strand > (i.e., one strand has one polh and one p10 and another strand > has one polh and one p10) "to minimize recombination" according > to the company. Promoters are ~ 70 and 90 bp. > > More than that, I might need to express two of the proteins > as fusions with the same small protein (162 bp). There is no > problem placing those on different strand. > > Because I don't really know much about homologous > recombination in either system, I worry if there are caveats > related to recombination and resulting instability of the > 1) transfer vector in E.coli 2) resulting baculoviral DNA > in the insect cells. > > Please give opinions on potential problems with the approach. > I always, of course, can go the route of co-infectin with three > separate viruses, painful as it is. > > Thanks, > > Dima > There was an article in Nature Methods some time ago that might answer some of your questions. The authors describe a baculovirus system suitable for the expression of whole protein complexes from one virus. This system is based on Invitrogens BacToBac-System but was modified so multiple transfer vectors can be integrated into the the bacmid harbouring the baculovirus genome. While there seems little probles associated with recombination in E.coli, even within the large baculovirus genome, the authors describe some problems with the stability of viruses if they are repeatedly overamplified. If this does not work out, you could always generate an individual baculovirus expressing each protein alone and coinfect cells with the same MOI. Best wishes Kay @article{ Author = {Fitzgerald, D. J. and Berger, P. and Schaffitzel, C. and Yamada, K. and Richmond, T. J. and Berger, I.}, Title = {Protein complex expression by using multigene baculoviral vectors}, Journal = {Nat Methods}, Volume = {3}, Number = {12}, Pages = {1021-32}, Note = {1548-7091 (Print) Journal Article Research Support, Non-U.S. Gov't}, Abstract = {Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression. Here we describe new transfer vectors and a combination of DNA recombination-based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.}, Year = {2006} } From olivaresn from niaid.nih.gov Mon Mar 3 12:43:20 2008 From: olivaresn from niaid.nih.gov (Olivares-Zavaleta, Norma (NIH/NIAID) [F]) Date: Mon Mar 3 13:30:20 2008 Subject: Proliferation Assay (Human T cells) Message-ID: <0FEED6FB2F3CF24384F2711FD61CC002092E92@NIHCESMLBX16.nih.gov> Hi everybody, I wonder if somebody could share some tips about Proliferation assay with human cells. I have the protocol for loading the cells with the CFSE reagent but I'd like to know more details about: 1) How many cells (CFSE loaded) to use? 2) How much time of incubation? I'll appreciate any comment at this respect. Norma From pow.joshi from gmail.com Mon Mar 3 19:04:34 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Mar 3 23:30:40 2008 Subject: Albumin depletion from sheep plasma In-Reply-To: References: Message-ID: <710764ea0803031604h1235fb6fh25bb826658dceff@mail.gmail.com> On 03/03/2008, Arianmanesh, Mitra wrote: > > Dears > > I need to deplete Albumin from sheep plasma while there is no defined > and perfect method for it. Actually, I used several methods for that > such as Immunoprecipitation and SwellGel Blue Albumin Removal Kit but I > could not get good results. So, now I need to know that how to deplete > Abumin from the sheep plasma. Can anybody give me an advise to do that yes, that is a tough one.. Perhaps, you could just use Blue sepharose CL-6B (trade name I think is Cibacron), and run your plasma one it as a column a few times or perhaps do a serial culk incubation. Now, if you wish to purify antibodies, you could use the afffinity columns; if you don't care about having or not having antibodies in the plasma/serum, you could probably use an ion exchanger as well. ...actually, I think I am a bit fuzzy here, and can be able to help you better if you let us know what exactly you wish to get out of the plasma. best Pow well? > > Kind regards, > > Mitra > > > > The University of Aberdeen is a charity registered in Scotland, No > SC013683. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From r06ma6 from abdn.ac.uk Tue Mar 4 05:07:54 2008 From: r06ma6 from abdn.ac.uk (Arianmanesh, Mitra) Date: Tue Mar 4 11:54:41 2008 Subject: Albumin depletion from sheep plasma Message-ID: Dear Pow Actually, I am doing Albumin depletion as I need the plasma for running the 2-D gel. if there is Albumin in the plasma, the gained 2-D gel would be very fussy and smeary due to lots of loaded Albumin as it covers other proteins on the 2-D gel. Now I'm trying Zoom fractionator for separating Albumin based on its PH, however it was not successful so far. I would be happy if you advise me. Sincerely, Mitra ________________________________ From: Pow Joshi [mailto:pow.joshi@gmail.com] Sent: 04 March 2008 00:05 To: Arianmanesh, Mitra Cc: methods@magpie.bio.indiana.edu Subject: Re: Albumin depletion from sheep plasma On 03/03/2008, Arianmanesh, Mitra wrote: Dears I need to deplete Albumin from sheep plasma while there is no defined and perfect method for it. Actually, I used several methods for that such as Immunoprecipitation and SwellGel Blue Albumin Removal Kit but I could not get good results. So, now I need to know that how to deplete Abumin from the sheep plasma. Can anybody give me an advise to do that yes, that is a tough one.. Perhaps, you could just use Blue sepharose CL-6B (trade name I think is Cibacron), and run your plasma one it as a column a few times or perhaps do a serial culk incubation. Now, if you wish to purify antibodies, you could use the afffinity columns; if you don't care about having or not having antibodies in the plasma/serum, you could probably use an ion exchanger as well. ...actually, I think I am a bit fuzzy here, and can be able to help you better if you let us know what exactly you wish to get out of the plasma. best Pow well? Kind regards, Mitra The University of Aberdeen is a charity registered in Scotland, No SC013683. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods The University of Aberdeen is a charity registered in Scotland, No SC013683. From pow.joshi from gmail.com Tue Mar 4 11:24:31 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Mar 4 11:55:13 2008 Subject: Albumin depletion from sheep plasma In-Reply-To: References: Message-ID: <710764ea0803040824u6946969fk3f6aa918bdf1a3b7@mail.gmail.com> On 04/03/2008, Arianmanesh, Mitra wrote: > > Dear Pow > > Actually, I am doing Albumin depletion as I need the plasma for running > the 2-D gel. if there is Albumin in the plasma, the gained 2-D gel would be > very fussy and smeary due to lots of loaded Albumin as it covers other > proteins on the 2-D gel. Now I'm trying Zoom fractionator for separating > Albumin based on its PH, however it was not successful so far. I would be > happy if you advise me. > > Sincerely, > I can only suggest that you do partial fractionation say starting with ammmonium sulphate cut offs and then ion exchangers, if the Cibacron blue does'nt work for you. These are some really ancient protocols but woork well. I understand the absolute frustration when there's this big blob of albumin sitting messing up an entire gel run.....esp. if you're looking for really low concentration species. hope that helps Pow Mitra > ** > > ------------------------------ > *From:* Pow Joshi [mailto:pow.joshi@gmail.com] > *Sent:* 04 March 2008 00:05 > *To:* Arianmanesh, Mitra > *Cc:* methods@magpie.bio.indiana.edu > *Subject:* Re: Albumin depletion from sheep plasma > > > > On 03/03/2008, Arianmanesh, Mitra wrote: > > > > Dears > > > > I need to deplete Albumin from sheep plasma while there is no defined > > and perfect method for it. Actually, I used several methods for that > > such as Immunoprecipitation and SwellGel Blue Albumin Removal Kit but I > > could not get good results. So, now I need to know that how to deplete > > Abumin from the sheep plasma. Can anybody give me an advise to do that > > > > yes, that is a tough one.. Perhaps, you could just use Blue sepharose > CL-6B (trade name I think is Cibacron), and run your plasma one it as a > column a few times or perhaps do a serial culk incubation. > Now, if you wish to purify antibodies, you could use the afffinity > columns; if you don't care about having or not having antibodies in the > plasma/serum, you could probably use an ion exchanger as well. ...actually, > I think I am a bit fuzzy here, and can be able to help you better if you let > us know what exactly you wish to get out of the plasma. > > best > Pow > > well? > > > > Kind regards, > > > > Mitra > > > > > > > > The University of Aberdeen is a charity registered in Scotland, No > > SC013683. > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > The University of Aberdeen is a charity registered in Scotland, No > SC013683. > From elnigm2003 from yahoo.com Mon Mar 3 19:05:01 2008 From: elnigm2003 from yahoo.com (Amr Negm) Date: Tue Mar 4 12:12:14 2008 Subject: bacillus subtilis expression protocol Message-ID: <745596.53494.qm@web55612.mail.re4.yahoo.com> i am ph.D. student and i am working with recombinant production of some enzymes by bacillus subtilis i have some transformed bacteria and i have tried someprotocoles for expression but it gives me no results and so i would be thankful if you help me and provide me with some ideas about this topic Amr Negm Amr Ali Negm Biochemistry and Molecular biology Institute Universitatsklinikum Hamburg-Eppendorf Hamburg University Mobile: +4917664188016 (mobile) Lab: +49 (0)40428 032387 Fax:+49 (0)40428 032904 http://www.chemie.uni-hamburg.de/bc/betzel/mitarbeiter/index.html --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. From ucgatan from ucl.ac.uk Tue Mar 4 11:55:26 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Tue Mar 4 15:16:04 2008 Subject: Debate In-Reply-To: <1o1yj.11887$0M3.7024@newsfe17.lga> References: <1o1yj.11887$0M3.7024@newsfe17.lga> Message-ID: On Sat, 1 Mar 2008, Aawara Chowdhury wrote: > In , > DK wrote: > > > One thing I am confused about: since its substrate is > > in the nucleus, you'd expect it to be targeted to nucleus? > > And yet, the SNAP is a full lenght human cDNA and the > > company provides a special SNAP plasmid that targets > > the tag to nucleus. How come? Don't all enzymes that > > work on chromosomes tend to localize to nucleus? > > Those are good observations and questions. I haven't used the tag, > but here's the information that I had received earlier. The SNAP tag > is a modified version of 6-O-methylguanine DNA methyltransferase, that > only recognizes para-substituted benzyl guanines. This substrate is > not recognized by the parental enzyme (and it is claimed, other cellular > proteins). > > The substrate (para-substituted benzyl guanine) is membrane permeable, > so it gets into cells very easily, and by the reaction you described, > modifies the SNAP tag with the para-substituent (which is typically > a fluorophore). > > Since nucleotides/nucleosides are both cytoplasmic and nuclear, > presumably the substrate goes to where the SNAP-tagged protein is. > > My concern is the same as yours. Does the tagged protein get dragged > to the nucleus? >From their document 'Wild-type AGT does not give any back-ground when labeling SNAP-tag fusion proteins in mammalian cells' [1]: "Wild-type AGT contains a DNA binding domain, resulting in a predominant nuclear localization, and reacts preferentially with DNA-bound alkyl guanine. Even though it is derived from human AGT, SNAP-tag is highly modified and optimized for use as a self-labeling tag. DNA-binding is abolished, leading to uniform distribution of SNAP-tag throughout the cytosol and the nucleus, and it is optimized to react rapidly with its free benzyl guanine and pyrimidyl guanine substrates." They say they've deleted the DNA-binding domain, so no nuclear localisation. DK, are you sure it's a full-length cDNA? tom [1] http://www.covalys.com/fileadmin/documents/SNAP_cell_no_wtAGT_labeling_visible.pdf -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From ucgatan from ucl.ac.uk Tue Mar 4 12:01:47 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Tue Mar 4 15:16:09 2008 Subject: Debate In-Reply-To: References: Message-ID: On Fri, 29 Feb 2008, DK wrote: > In article , Tom Anderson wrote: > > >Note that in the case of some fluorescent proteins, there's also a risk of > >oligomerisation artifacts: wildtype FPs (all or just some) are tetrameric > > Happily, not the classic Aequorea GFP. That one only forms weak dimers > that are detectable only at high concentrations. Even in many crystals > such a dimer is not observed. Ah! I didn't realise that, thanks and apologies. > >There is a relatively new alternative to GFP for fluorescent tagging of > >proteins in live cells in the shape of the FlAsH/ReAsH system: > > > >http://www.invitrogen.com/content.cfm?pageid=11922 > > > >Basically, you put a magic tag on your protein, > > The magic tag with two exposed Cys is a subject to many crosslinking > artifacts. Probably not a problem for most cytoplasmic proteins > (reducing environment) but must be a huge problem for anything that goes > through secretion pathway. Yes, i assume it'd be a complete non-starter for anyhing outside the cytoplasm. tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From ucgatan from ucl.ac.uk Tue Mar 4 12:00:23 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Tue Mar 4 15:16:16 2008 Subject: Debate In-Reply-To: References: Message-ID: On Fri, 29 Feb 2008, DK wrote: > In article , aawara@pontiff-playground.org wrote: > >In , > > DK wrote: > > > >>>There is a relatively new alternative to GFP for fluorescent tagging of > >>>proteins in live cells in the shape of the FlAsH/ReAsH system: > >>> > >>>http://www.invitrogen.com/content.cfm?pageid=11922 > >>> > >>>Basically, you put a magic tag on your protein, > >> > >> The magic tag with four exposed Cys is a subject to many > >> crosslinking artifacts. Probably not a problem for most > >> cytoplasmic proteins (reducing environment) but must > >> be a huge problem for anything that goes through secretion > >> pathway. > > > >Any thoughts on the SNAP tag? > >http://www.covalys.com/products/snap-cell-labeling-in-cells.html > > Interesting! I did not know about it, thanks. > > Since the company does not seem to provide any concrete > details, I had to download their plasmid sequence to BLAST > and find out what "SNAP tag" is. > > Human 6-O-methylguanine DNA methyltransferase. > Only ~ 20K, good, no tight dimers in the crystal, termini are > not involved in the active site, so presumably works on both > N- and -C end. The question what a ligand is and how > tightly it binds seems to be answered by PDB code 1YFH : > > "Involved in the cellular defense against the biological effects of > o6-methylguanine (o6-meg) in dna. This is a suicide reaction: > the enzyme is irreversibly inactivated. Repairs alkylated > guanine in dna by stoichiometrically transferring the alkyl > group at the o-6 position to a cysteine residue in the enzyme." > > E.g., the binding is irreversible. > > All in all, seems to be appealing. I am contemplating some > in vitro FRET and it maybe just what doctor ordered. So what advantage over GFP and friends? 20 kDa is the same ballpark as GFP, and we know that GFP has good monomericity and fusability. I guess with SNAP you can use a chemical fluorophore, which can be much brighter and more stable. And of course, you get to choose the colour *after* you've expressed the protein, which is nice! Is there a solution for two-colour SNAP? You'd need another modified version of the enzyme that only recognised some other form of substituted nucleotide, i suppose. tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From R.Jayakumar from roswellpark.org Tue Mar 4 16:20:03 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Mar 4 19:06:45 2008 Subject: bacillus subtilis expression protocol In-Reply-To: <745596.53494.qm@web55612.mail.re4.yahoo.com> References: <745596.53494.qm@web55612.mail.re4.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E79FC@VISTA.roswellpark.org> Expression of what enzyme and is the transformed bacteria also Bacillus? What kind of vector did you use for expression? Where is the cDNA or gene from (if bacteria, what bacteria)? What is the promoter you are using to express this gene? What kind of transformation did you do? Have you verified for the presence of the plasmid in the transformed bacteria? When posting questions, it will be really helpful if you can give a detailed outline of your experiment. This will help people to respond quicker. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Amr Negm Sent: Monday, March 03, 2008 7:05 PM To: methods@magpie.bio.indiana.edu Subject: bacillus subtilis expression protocol i am ph.D. student and i am working with recombinant production of some enzymes by bacillus subtilis i have some transformed bacteria and i have tried someprotocoles for expression but it gives me no results and so i would be thankful if you help me and provide me with some ideas about this topic Amr Negm Amr Ali Negm Biochemistry and Molecular biology Institute Universitatsklinikum Hamburg-Eppendorf Hamburg University Mobile: +4917664188016 (mobile) Lab: +49 (0)40428 032387 Fax:+49 (0)40428 032904 http://www.chemie.uni-hamburg.de/bc/betzel/mitarbeiter/index.html --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From meltonbf2007 from tragoc.f2s.com Tue Mar 4 18:15:43 2008 From: meltonbf2007 from tragoc.f2s.com (Clive Tregaskes) Date: Tue Mar 4 20:10:17 2008 Subject: solid phase colony growth Message-ID: <1oydnfZQR44FS1DanZ2dnUVZ8smgnZ2d@pipex.net> Hi all, some time ago there was a paper (I think it was a NAR methods paper) regarding growing bacteria for cDNA libraries etc. in a semi-solid gel rather than on plates so (as the authors claimed) the colonies all grew to the same size and you didn't lose representation. Anyone have any idea where this paper came from- I've tried various searches and I can't find it again- otherwise anybody got the method- Its something simple like suspending the cells in LB/1% low melting point agar but not certain and can't remember how you get the bacteria back out again all help gratefully appreciated Clive From alicia.navetta from ucsf.edu Wed Mar 5 17:54:54 2008 From: alicia.navetta from ucsf.edu (Alicia Navetta) Date: Wed Mar 5 18:54:03 2008 Subject: Cloning problems with nls-lacZ Message-ID: <9C1CEB61-6123-40E4-97D0-F962E22B2F53@ucsf.edu> Hi, Just a shot in the dark, but I'm having exactly the same problem cloning nuclear-LacZ into a plasmid. Did you ever find a resolution. Thanks, Alicia Alicia Navetta Staff Research Associate I Cardiovascular Research Institute University of California, San Francisco UCSF MC 2711 1550 4th Street Rock Hall, Room 382 San Francisco, CA 94158-2324 (415) 476-3231 alicia.navetta@ucsf.edu From diana_barbosa from sapo.pt Thu Mar 6 12:51:51 2008 From: diana_barbosa from sapo.pt (diana_barbosa@sapo.pt) Date: Thu Mar 6 13:24:34 2008 Subject: Triton X-100 Message-ID: <20080306175151.mqok31j5cowwgssw@w9.mail.sapo.pt> Hello, I'm having some trouble with the solubilization of proteins contained in a lipidic secretion we're studying (using 2DE). To overcome this problem, I'm preparing a series of trials with different lysis buffers, prepared with detergents alternative/complementary to CHAPS. One of the alternatives mentioned in the literature is Triton X-100, but in some papers it appears that this detergent doesn't help solubilize hydrophobic proteins but rather precipitates them, creating a 2 fractions (hydrophilic and hydrophobic proteins). Is this true or am I misinterpreting? Any help will be very welcome. Thank you, Diana From allison from nospam.com Thu Mar 6 15:27:52 2008 From: allison from nospam.com (allisonh) Date: Thu Mar 6 18:13:51 2008 Subject: homemade ECL Message-ID: <47d04ed4$0$16009$9a6e19ea@news.newshosting.com> While digging through some old papers I found a newsgroup posting with a recipe for ECL reagents (posting was from 1998 so practically the dark ages in internet years). Is anyone using homemade ECL currently? I am wondering if it is worth my time to order ingredients and try it out or will I be disappointed by the sensitivity. TIA Allison From legatek from hotmail.com Thu Mar 6 16:04:27 2008 From: legatek from hotmail.com (Kyle Legate) Date: Thu Mar 6 18:13:55 2008 Subject: Triton X-100 In-Reply-To: References: Message-ID: <63b4ihF26dq1uU1@mid.individual.net> diana_barbosa@sapo.pt wrote: > Hello, > > I'm having some trouble with the solubilization of proteins contained in > a lipidic secretion we're studying (using 2DE). > To overcome this problem, I'm preparing a series of trials with > different lysis buffers, prepared with detergents > alternative/complementary to CHAPS. > One of the alternatives mentioned in the literature is Triton X-100, but > in some papers it appears that this detergent doesn't help solubilize > hydrophobic proteins but rather precipitates them, creating a 2 > fractions (hydrophilic and hydrophobic proteins). Is this true or am I > misinterpreting? > > Any help will be very welcome. > Thank you, > > Diana > I think you mean Triton X-114, which is used for cloud point assays. http://www.ncbi.nlm.nih.gov/pubmed/6257680?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum From pilamca from yahoo.ca Thu Mar 6 18:53:30 2008 From: pilamca from yahoo.ca (lam) Date: Thu Mar 6 23:48:47 2008 Subject: TAC (IL2R-alpha) chimera In-Reply-To: <20080306175151.mqok31j5cowwgssw@w9.mail.sapo.pt> Message-ID: <967161.48359.qm@web51108.mail.re2.yahoo.com> Hello, I have made a chimera of the IL-2R and C-terminal tail of my protein to check for sorting signal. I am wondering if the last 13 amino acid tail in the IL-2R would be important in the design of this construct? Does it affect trafficking. Thank-you. Sincerely Ping Looking for the perfect gift? Give the gift of Flickr! http://www.flickr.com/gift/ From arnec from bio.umass.edu Fri Mar 7 12:46:43 2008 From: arnec from bio.umass.edu (arnec@bio.umass.edu) Date: Fri Mar 7 15:45:32 2008 Subject: Film Developer Message-ID: <2748.159.189.36.78.1204912003.squirrel@www.bio.umass.edu> I am in the market for a film developer that is dependable and conservative with dev/fix fluids (~$4,500). Can anybody recommend one (no vendors please)? I have had a bad experience with AlphaTek, and I have been recommended a Konica Milota SRX-101A. I am also open to CCD camera based detection of ECL OR building my own, if somebody can recommend a good source of information on doing so. My appologies if this is the wrong forum to place such an inquiry. Thank you. From arnec from bio.umass.edu Fri Mar 7 13:13:52 2008 From: arnec from bio.umass.edu (arnec@bio.umass.edu) Date: Fri Mar 7 15:46:01 2008 Subject: Film developer Message-ID: <2822.159.189.36.78.1204913632.squirrel@www.bio.umass.edu> I am in the market for a film developer that is dependable and conservative with dev/fix fluids (~$4,500). Can anybody recommend one (no vendors please)? I have had a bad experience with AlphaTek, and I have been recommended a Konica Milota SRX-101A. I am also open to CCD camera based detection of ECL OR building my own, if somebody can recommend a good source of information on doing so. My appologies if this is the wrong forum to place such an inquiry. Thank you. From legatek from hotmail.com Fri Mar 7 12:39:37 2008 From: legatek from hotmail.com (Kyle Legate) Date: Fri Mar 7 15:46:09 2008 Subject: Triton X-100 In-Reply-To: References: <63b4ihF26dq1uU1@mid.individual.net> Message-ID: <63dcucF26tso3U1@mid.individual.net> DK wrote: > > 2. Even after filtering out the obvious insoluble light lipid-like > fraction, the resulting TX-100 lysates have almost magical > ability to clog columns. With large scale/concentrated > lysate, it's very quickly getting to a point where the column > won't flow by gravity at all. None of that ever happens with > E.coli lysates. I always (perhaps naively) interpreted this > as "cholesterol-rich lipid-protein complexes are not soluble > in TX-100 and get aggregated/concentrated instead, binding > to everything and cloging pores in agarose". > Insolubility in TX-100 is one of the hallmarks of so-called "lipid rafts", and very well might be the cause of clogged columns, but if the lipidic secretion the OP is working with is low in cholesterol, TX-100 should definitely be considered in the screen. If the secretion is cholesterol rich, adding some digitonin might help to solubilize it. > Octyl-glucoside would be my first choice to mix with > CHAPS if I must mix CHAPS with something. In some cases, > we also had pretty good results with LDAO (in terms of > rescuing difficult proteins, no 2DE). > From ebs15242 from creighton.edu Fri Mar 7 15:19:59 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Fri Mar 7 15:46:15 2008 Subject: embryo powder Message-ID: <47D1A36F.7080600@creighton.edu> We're going to be making some embryo powder for antibody preabsorption for in situ hybridization. Pretty much as per this protocol: http://www.oup.co.uk/pdf/pas/12-4-5.pdf We're wondering whether it's ok to pulverize the embryos in liquid nitrogen before homogenization in PBS. I'm not sure our homogenizer would be able to handle whole embryos. That's not going to affect the antigenicity of the embryo powder, is it? From josenet from tiscali.co.uk Fri Mar 7 22:52:26 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Mar 7 23:54:56 2008 Subject: homemade ECL References: <47d04ed4$0$16009$9a6e19ea@news.newshosting.com> <6n2Aj.486$9b1.425@newsfe06.lga> Message-ID: <63egs3F270vvmU1@mid.individual.net> "DK" wrote in message news:6n2Aj.486$9b1.425@newsfe06.lga... > In article <47d04ed4$0$16009$9a6e19ea@news.newshosting.com>, allisonh > wrote: > >>Is anyone using homemade ECL currently? I am >>wondering if it is worth my time to order ingredients and try it out or >>will I be disappointed by the sensitivity. > > I remember trying it at around this time and comparing it back to > back with Pierce's "SuperSignal" and concluding that, for as > long as I can't make home-made Pierce version, the trouble of > making traditional ECL is not worth it if you want sensitivity and > peace of mind every time you do Western where you don't > exactly know what kind of signal to expect. > > Truth is, however, I was never satisfied with Amersham's original > ECL mix either. > > DK We've been using a homemade version for years now. Pretty cheap and easy. Out of my head, I just add 670ul of 1.5M Tris pH8.8 and water to 10ml. Then we have frozen aliquots of luminol and p-coumaric acid (i forget the amounts/concentrations... can you tell I just do it mechanically now?)... all we do is add one aliquot of each, mix well, add 3-4ul of H2O2, and pour over the membrane. I don't do a lot of westerns, but others in my lab do and noone complained about sensitivity when we stopped buying the commercial version. The one thing that is different, is that our homemade version goes off a lot more quickly. I think that after 15 minutes or so you won't get much. But often we only need to expose for a minute or so to get good signals (of course, thsi is highly dependant on the antibody and what you're actually after)... My "routine" tends to be a quick exposure (20-30 seconds) and expose another film while developing the first. It takes about 2min for the film to come out. Then depending on what i see, either I develop the one I've been exposing (2min already) or leave it a bit longer. You do have to work a bit faster than with commercial ECL, but it works quite well once you get the hang of it. If interested I can post our recipe. The ingredients are cheap and they'll last a looong time. Jose From aawara from pontiff-playground.org Sat Mar 8 05:52:48 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat Mar 8 16:00:03 2008 Subject: homemade ECL References: <47d04ed4$0$16009$9a6e19ea@news.newshosting.com> <6n2Aj.486$9b1.425@newsfe06.lga> <63egs3F270vvmU1@mid.individual.net> Message-ID: <4euAj.16973$097.13453@newsfe21.lga> In <63egs3F270vvmU1@mid.individual.net>, Jose de las Heras wrote: > I don't do a lot of westerns, but others in my lab do and noone complained > about sensitivity when we stopped buying the commercial version. The Pierce Supersignal reagents that Dima referred to are far more sensitive than Amersham's ECL (ECL plus) reagents. We also use a home-made ECL reagent that is similar to yours, and the sensitivity of our reagent is similar to Amersham's ECL reagent (or Pierce's ECL reagent). The Supersignal reagents permit detection of 10 - 50 femtograms of protein. In practical terms this means that if we use a particular antibody at a 1:1000 dilution with our home-made ECL reagent, we can use it at 1:15000 with the Supersignal Femto reagent. Useful for some Abs that are very expensive. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From arnec from bio.umass.edu Sat Mar 8 12:29:47 2008 From: arnec from bio.umass.edu (arnec@bio.umass.edu) Date: Sat Mar 8 16:00:16 2008 Subject: homemade ECL In-Reply-To: <63egs3F270vvmU1@mid.individual.net> References: <47d04ed4$0$16009$9a6e19ea@news.newshosting.com> <6n2Aj.486$9b1.425@newsfe06.lga> <63egs3F270vvmU1@mid.individual.net> Message-ID: <54192.128.119.55.30.1204997387.squirrel@www.bio.umass.edu> I regularly do western blots with a method similar to the one described by Jose. It works well for standard western blots. In my experience it is not as sensitive as commercially available reagents such as Pierce Super Signal West Dura. This is sometimes a benefit however, b/c it may result in less background. If I'm working with a new antibody or sample type, I usually use the homemade reagents, and if greater sensitivity is required I move on to the commercial ($$$$$$$$) stuff. Stock sol'ns (50 ul aliquots) 1)90 mM coumaric acid 2)250 mM luminol Working sol'ns A) Add 22ul coumaric acid and 50ul luminol to 5mL cold 0.1M Tris pH8.5 B) Add 3ul H2O2 to 5mL cold 0.1M Tris pH8.5 Mix A) and B), add to blot for 1 min, continue with exposure to film etc. > > "DK" wrote in message > news:6n2Aj.486$9b1.425@newsfe06.lga... >> In article <47d04ed4$0$16009$9a6e19ea@news.newshosting.com>, allisonh >> wrote: >> >>>Is anyone using homemade ECL currently? I am >>>wondering if it is worth my time to order ingredients and try it out or >>>will I be disappointed by the sensitivity. >> >> I remember trying it at around this time and comparing it back to >> back with Pierce's "SuperSignal" and concluding that, for as >> long as I can't make home-made Pierce version, the trouble of >> making traditional ECL is not worth it if you want sensitivity and >> peace of mind every time you do Western where you don't >> exactly know what kind of signal to expect. >> >> Truth is, however, I was never satisfied with Amersham's original >> ECL mix either. >> >> DK > > We've been using a homemade version for years now. Pretty cheap and easy. > Out of my head, I just add 670ul of 1.5M Tris pH8.8 and water to 10ml. > Then > we have frozen aliquots of luminol and p-coumaric acid (i forget the > amounts/concentrations... can you tell I just do it mechanically now?)... > all we do is add one aliquot of each, mix well, add 3-4ul of H2O2, and > pour > over the membrane. > > I don't do a lot of westerns, but others in my lab do and noone complained > about sensitivity when we stopped buying the commercial version. The one > thing that is different, is that our homemade version goes off a lot more > quickly. I think that after 15 minutes or so you won't get much. But often > we only need to expose for a minute or so to get good signals (of course, > thsi is highly dependant on the antibody and what you're actually > after)... > My "routine" tends to be a quick exposure (20-30 seconds) and expose > another > film while developing the first. It takes about 2min for the film to come > out. Then depending on what i see, either I develop the one I've been > exposing (2min already) or leave it a bit longer. You do have to work a > bit > faster than with commercial ECL, but it works quite well once you get the > hang of it. > > If interested I can post our recipe. The ingredients are cheap and they'll > last a looong time. > > Jose > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From manhochu from gmail.com Sun Mar 9 15:59:57 2008 From: manhochu from gmail.com (Man-Ho Chu) Date: Sun Mar 9 19:42:39 2008 Subject: Truncated inserts Message-ID: <680c9f31-696f-4347-89f9-429e9df04e55@i7g2000prf.googlegroups.com> Hi everybody, I'm trying to get a 2.7 kb insert (excised from a parent plasmid) into a plasmid. The insert was excised w/ Nco I and Sma I. The vector was prepared by cutting w/ Eco RI, filling in w/ Pfu, and cutting w/ Nco I. The vetor was CIPed and the insert was cut from gel and purified. I've done the ligation a few times but the clones with insert that I sequenced always have the insert truncated at the 3' end, the length of the missing sequence ranging from 2 kb to 400 bp. I'm not sure whether I can get the full length insert by keep screening, and I can't figure out why this is happening. Does anyone have any idea what might have happened, and does anyone have any idea how to trouble shoot this problem? Any input is greatly appreciated! TIA! Man-Ho Chu From engelbert_buxbaum from hotmail.com Mon Mar 10 08:52:30 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Mar 10 11:42:39 2008 Subject: Triton X-100 References: <63b4ihF26dq1uU1@mid.individual.net> <63dcucF26tso3U1@mid.individual.net> Message-ID: Am 07.03.2008, 13:39 Uhr, schrieb Kyle Legate : > Insolubility in TX-100 is one of the hallmarks of so-called "lipid > rafts", and very well might be the cause of clogged columns, but if the > lipidic secretion the OP is working with is low in cholesterol, TX-100 > should definitely be considered in the screen. If the secretion is > cholesterol rich, adding some digitonin might help to solubilize it. I agree. One may also consider Tween detergents (properties similar to Triton) or the Elugents, which are sugar-alkane based industrial detergents (much cheaper than OG or DM). From borderline86 from yahoo.com Tue Mar 11 13:13:15 2008 From: borderline86 from yahoo.com (Maurizio Affer) Date: Tue Mar 11 13:23:07 2008 Subject: Embarssing problem Message-ID: <116511.42385.qm@web90503.mail.mud.yahoo.com> Hi there I'm trying to express a small peptide in E. coli. After doing my cloning in Dh5alpha using a pgex-6p1 vector (amersham) driven by a TAc prmoter I moved the plasmid into a Bl21-De3 strain. The problem is I discovered that I'm not even able to induce expression of GST (which is in the vector of course)! I induce at an OD of 0.8 with IPTG final of 0.1mM for 2 hours. All the trouble shooting are about "not enough protein induced", but here I'm at "no GST induction"! Please help whatever comes to your mind let me know... Maurizio Affer PhD Memorial Sloan-Kettering Institute Zuckerman Building Room 1731B New York (NY) 10065 Tel.646-888-2072 afferm@mskcc.org ____________________________________________________________________________________ Looking for last minute shopping deals? Find them fast with Yahoo! Search. http://tools.search.yahoo.com/newsearch/category.php?category=shopping From allison from nospam.com Tue Mar 11 12:53:27 2008 From: allison from nospam.com (allisonh) Date: Tue Mar 11 13:23:14 2008 Subject: homemade ECL In-Reply-To: <63egs3F270vvmU1@mid.individual.net> References: <47d04ed4$0$16009$9a6e19ea@news.newshosting.com> <6n2Aj.486$9b1.425@newsfe06.lga> <63egs3F270vvmU1@mid.individual.net> Message-ID: <47d6c210$0$17177$9a6e19ea@news.newshosting.com> Jose de las Heras wrote: > "DK" wrote in message > news:6n2Aj.486$9b1.425@newsfe06.lga... >> In article <47d04ed4$0$16009$9a6e19ea@news.newshosting.com>, allisonh >> wrote: >> >>> Is anyone using homemade ECL currently? I am >>> wondering if it is worth my time to order ingredients and try it out or >>> will I be disappointed by the sensitivity. >> I remember trying it at around this time and comparing it back to >> back with Pierce's "SuperSignal" and concluding that, for as >> long as I can't make home-made Pierce version, the trouble of >> making traditional ECL is not worth it if you want sensitivity and >> peace of mind every time you do Western where you don't >> exactly know what kind of signal to expect. >> >> Truth is, however, I was never satisfied with Amersham's original >> ECL mix either. >> >> DK > > We've been using a homemade version for years now. Pretty cheap and easy. > Out of my head, I just add 670ul of 1.5M Tris pH8.8 and water to 10ml. Then > we have frozen aliquots of luminol and p-coumaric acid (i forget the > amounts/concentrations... can you tell I just do it mechanically now?)... > all we do is add one aliquot of each, mix well, add 3-4ul of H2O2, and pour > over the membrane. > > I don't do a lot of westerns, but others in my lab do and noone complained > about sensitivity when we stopped buying the commercial version. The one > thing that is different, is that our homemade version goes off a lot more > quickly. I think that after 15 minutes or so you won't get much. But often > we only need to expose for a minute or so to get good signals (of course, > thsi is highly dependant on the antibody and what you're actually after)... > My "routine" tends to be a quick exposure (20-30 seconds) and expose another > film while developing the first. It takes about 2min for the film to come > out. Then depending on what i see, either I develop the one I've been > exposing (2min already) or leave it a bit longer. You do have to work a bit > faster than with commercial ECL, but it works quite well once you get the > hang of it. > > If interested I can post our recipe. The ingredients are cheap and they'll > last a looong time. > > Jose > > I'm using ECL plus right now and find that the sensitivity is fine for what I am doing. So I would be interested in trying a homemade version to save $$. One question though - since it is only active for 15 minutes, is it possible to 'reactivate' the blot by resoaking it in the reagent if the correct exposure is not done the first time thru? I'm thinking of the days when there is a lineup in the dark room or the like :) Also, could you please post your recipe (if it is different than the one that Arnec posted). Thanks a lot Allison From arnec from bio.umass.edu Tue Mar 11 16:25:45 2008 From: arnec from bio.umass.edu (arnec@bio.umass.edu) Date: Tue Mar 11 17:52:04 2008 Subject: homemade ECL Message-ID: <2163.159.189.36.78.1205270745.squirrel@www.bio.umass.edu> If you need to redo a detection, I rinse the blot in PBS, then put it back into secondary (which I would have saved) and continue from there. Also, in the protocol that I summarized previously, I the coumeric acid and luminol are dissolved in DMSO. Arne From kaj.stenberg from helsinki.fi.invalid Wed Mar 12 01:09:54 2008 From: kaj.stenberg from helsinki.fi.invalid (kaj.stenberg@helsinki.fi.invalid) Date: Wed Mar 12 12:44:12 2008 Subject: Embarssing problem References: Message-ID: Maurizio Affer wrote: > Hi there > I'm trying to express a small peptide in E. coli. > After doing my cloning in Dh5alpha using a pgex-6p1 > vector (amersham) driven by a TAc prmoter I moved the > plasmid into a Bl21-De3 strain. > The problem is I discovered that I'm not even able to > induce expression of GST (which is in the vector of > course)! > I induce at an OD of 0.8 with IPTG final of 0.1mM for > 2 hours. > All the trouble shooting are about "not enough protein > induced", but here I'm at "no GST induction"! > Please help whatever comes to your mind let me know... Have you tested different media/induction time, they can vary surprisingly much? For one project (using BL21-De3) I did some testing with the following results: Induction time (0.1mM IPTG at OD 0.8): 10% after 2h 90% after 15h, best at 20 h Using the long induction time, media/temperature: LB 37 degrees <5% LB 28 degrees 50% TB 37 degrees 100% TB 28 degrees 70% YT 37 degrees 10% YT 28 degrees 25% So although the protocol you use is the standard one the optimal parameters can vary a lot! -- ============================================================================= Kaj Stenberg, Ph. D Department of Biosciences tel. +358-9-191 59682 Division of Biochemistry fax +358-9-191 59068 P. O. Box 56, Viikinkaari 5 e-mail: kaj.stenberg@helsinki.fi FIN-00014 University of Helsinki Finland This message is environmentally friendly: every bit was created using 100% recycled electrons. ============================================================================= From prae from gmx.net Wed Mar 12 04:07:59 2008 From: prae from gmx.net (Christian Praetorius) Date: Wed Mar 12 12:44:43 2008 Subject: Embarssing problem References: Message-ID: <63pkraF28ltdkU1@mid.individual.net> Maurizio Affer wrote: >I induce at an OD of 0.8 with IPTG final of 0.1mM for >2 hours. This is a quite short time after induction and also a very small concentration for the IPTG. We ususally induce for 3-5 hours with 1mM IPTG final using pGEX and BL21. Christian -- X-no-Sig: yes From j.robson from imb.uq.edu.au Tue Mar 11 23:19:30 2008 From: j.robson from imb.uq.edu.au (Jonathan Robson) Date: Wed Mar 12 12:44:58 2008 Subject: mAb anti-mouse CD15 - wanted Message-ID: <000e01c883f8$4494a720$32756682@IMBPC.AD> Did u find any mouse CD15? I am also interested in purchasing some but am having difficulty finding any. ------------=_47D759DD.AD057848-- From metugenetics from yahoo.com Wed Mar 12 13:59:32 2008 From: metugenetics from yahoo.com (Ozan Aygun) Date: Wed Mar 12 18:29:59 2008 Subject: Embarssing problem Message-ID: <21669.10253.qm@web90406.mail.mud.yahoo.com> Hi there, I am routinely using the same vector. I reccomend you to use IPTG to a final of 1mM. Moreover, start inducing at OD600: 0.2. Leave temperature at 37 C, whic is OK to see only GST expression. Worth to keep some Glucose before induction. May worth to replace or re-prepare your IPTG stock as well. (In Bl21-DE-RIL strain there is an internal reporter plasmid that shows you up on the coomassie stained gel when the IPTG induction indeed works). If nothing works, then you may better to sequence the promoter and operator sequences of your backbone vector to check any modification from the original one. Good luck! Ozan ____________________________________________________________________________________ Never miss a thing. Make Yahoo your home page. http://www.yahoo.com/r/hs From novalidaddress from nurfuerspam.de Thu Mar 13 02:53:55 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Mar 13 12:45:15 2008 Subject: Embarssing problem References: Message-ID: <09951c57-925a-4794-b83b-1d63cd5f8cac@e60g2000hsh.googlegroups.com> Hi Maurizio, if your constructs and bacteria are ok, then you just may try various conditions. This is called RE-search. Not embarrassing at all. Best reagrds, Wo From sgdalvi from gmail.com Fri Mar 14 00:57:47 2008 From: sgdalvi from gmail.com (Sunil Dalvi) Date: Fri Mar 14 09:48:24 2008 Subject: colony PCR Message-ID: I am in need of colony PCR method . I got the positive colonies for Agro but not getting the band for insert. I got the band for positive control in the same reaction. where would be the problem -- S.G.Dalvi, Vasantdada Sugar Institute,Pune Manjari bk, India http://www.vsisugar.com/ From e_barouki from hotmail.com Fri Mar 14 08:19:52 2008 From: e_barouki from hotmail.com (Emad) Date: Fri Mar 14 09:48:37 2008 Subject: Dam and Dcm methylation problem Message-ID: Hello Can somebody tell me if I can use the XL1 or NEB5 alpha E coli for cloning plasmids which will be used for digestion by enzymes that are sensitive to Dam, Dcm and CG methylation. I know that these strains are not Dcm- ,Dam- strains. However can I demethylate my isolated plasmid later on, for further enzymatic digestion and how? Thank in Advance Emad From mlsulliv from wisc.edu Fri Mar 14 10:23:20 2008 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Fri Mar 14 12:17:17 2008 Subject: colony PCR In-Reply-To: References: Message-ID: <5AAC050A-EE47-4089-BE9F-974FFF4455C5@wisc.edu> To do PCR on agro, I usually grow up a liquid culture. Harvest 100 ul of culture by centrifugation, resuspend in 20 ul water, then boil for 10 min. Spin out debris for 5 min. I use 5 ul of this in a 25 ul PCR reaction (which is probably a lot more than needed). This procedure works well for me for detecting genes in the Ti-plasmid. Usually for checking my binary construct I do a miniprep and check the digestion pattern. I prefer this since it gives you more information than just amplifying a portion of a gene (i.e. digests might tell you you've had a rearrangement, etc.). I've had good luck doing Qiagen minipreps with agrobacterium. Quality of the DNA is usually good enough to work in a restriction digestion. I've had more variable results with standard minipreps: digestion with these sometimes works, sometimes doesn't. If you make DNA from agro and your digests don't work, the DNA is usually good enough to retransform into E. coli. Plasimd can then be prepped from E. coli for analysis. One thing to keep in mind with DNA from agrobacterium is that methylation is different and may effect some restriction sites with overlapping Dam methylation (e.g. ClaI). Doing restriction digests may take a bit longer than doing a quick PCR reaction, but compared to how much time will be spent on plant transformation, I think it's worth spending a few minutes to make sure the the binary plasmid in your agro is correct. Mike On Mar 14, 2008, at 12:57 AM, Sunil Dalvi wrote: > I am in need of colony PCR method . I got the positive colonies for > Agro but > not getting the band for insert. I got the band for positive > control in the > same reaction. > where would be the problem > > -- > S.G.Dalvi, > Vasantdada Sugar Institute,Pune > Manjari bk, India > http://www.vsisugar.com/ > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods --- Michael L. Sullivan Plant Research Molecular Geneticist US Dairy Forage Research Center ARS-USDA 1925 Linden Drive West Madison, WI 53706 (608) 890-0046 (Phone) (608) 890-0076 (FAX) From carla.beukes from gmail.com Sat Mar 15 03:37:03 2008 From: carla.beukes from gmail.com (Carla Beukes) Date: Sat Mar 15 19:19:28 2008 Subject: Digestion Problem Message-ID: I am currently working with the Beta 2 adrenergic receptor polymorphims, Arg16Gly and Glu27Gly. The PCR seems to be working fine and I get product but after I have digested with the enzymes NCoI or BbvI nothing shows up on the gel. It is completely blank. Any suggestions as to what I may be doing wrong? Carla Beukes (carla.beukes@gmail.com) From analia_alet from intech.gov.ar Sat Mar 15 08:29:00 2008 From: analia_alet from intech.gov.ar (Analia Alet) Date: Sat Mar 15 19:19:40 2008 Subject: colony PCR In-Reply-To: <200803141704.m2EH4bL14772@net.bio.net> References: <200803141704.m2EH4bL14772@net.bio.net> Message-ID: <1205587740.47dbcf1c62fa5@webmail.intech.gov.ar> Hi Sunil You can find a good method for colony PCR at Sambrook et al. (Manniatis) Also what I used to do was to start the PCR without Taq. After a first cycle of 5 min 91?C, I stopped it manually and added Taq. Then I continued the PCR as usual. This first cycle is important to disrupt the cells. Another advice is to put very but very little bacterial colony into the PCR tube. Just touch the colony, don't worry if you don't see the bacterias at the end of the tip (maybe that's the problem; perhaps you are placing half the entire colony into the PCR tube) Finally, my PCR protocol (but it depends on your primers) 1 cycle 91?C 5 min x1 stop manually, place in ice and add Taq 2 cycle (x35) 94?C 1 min; 60?C 1 min; 72?C 1 min 3 cycle 72?C 5 min x1 4 hold 4?C Hope it works for you best regards Anal?a >I am in need of colony PCR method . I got the positive colonies for Agro but >not getting the band for insert. I got the band for positive control in the >same reaction. >where would be the problem From novalidaddress from nurfuerspam.de Sat Mar 15 20:45:00 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sun Mar 16 12:36:50 2008 Subject: Digestion Problem References: Message-ID: <04e4c322-b906-4632-b730-288db2f14131@c33g2000hsd.googlegroups.com> Hi Carla, if you get a band with your PCR, you can see the marker on the gel but there is no signal after the digest, you most likely have a DNAse somewhere in your system. Be sure to include sufficient and suitable controls for the digest (in your case, PCR product on the gel should be enough). To locate the possible source of DNA contamination, you might want to leave out *one* component of the digest at a time. Maybe it's more straightforward to get everything new (water, buffer) than to try around for a long time. HTH Wo From M.Dunowska from massey.ac.nz Sun Mar 16 04:49:48 2008 From: M.Dunowska from massey.ac.nz (Dunowska, Magda) Date: Sun Mar 16 12:37:07 2008 Subject: "freeze and squeeze" DNA extraction Message-ID: Hi, Sorry to revisit the subject - I have just gone through the archives and can't find a description of a method I used to use a few years ago to get DNA from agarose gels. I used normal (not LMP) agarose and TBE buffer. All I remember is that it involved freezing and thawing a gel slice, but I think I was also adding water or TE (can't remember what and how much), heating the thawed gel to either 65 or 70 degrees for 5 or 10 minutes (?) and mashing it all up with the blue tip before centrifuging everything at 4 degrees to collect supernatant. I just got back to the molecular work after a few years in a slightly different role, and (surprisingly...) can't recall all the details of procedures that I used to do on a daily basis and never bothered to write down outside of the lab books that are no longer with me...Anybody out there who uses/used a similar variant of the "freeze and squeeze" and can give me the details? Thanks! Magda From elnigm2003 from yahoo.com Sat Mar 15 19:29:13 2008 From: elnigm2003 from yahoo.com (Amr Negm) Date: Sun Mar 16 12:39:30 2008 Subject: colony PCR method for Bacillus subtilis Message-ID: <136023.98989.qm@web55615.mail.re4.yahoo.com> I am in need of colony PCR method for Bacillus subtilis. I got the positive colonies for but i can not make this PCR waiting for help Amr --------------------------------- Looking for last minute shopping deals? Find them fast with Yahoo! Search. From siheng.xiang from gmail.com Sun Mar 16 07:35:02 2008 From: siheng.xiang from gmail.com (kalva) Date: Sun Mar 16 12:39:44 2008 Subject: "Triton"s Message-ID: when extract proteins from membrane, usually we try Triton X100 first, but there are different kinds of Tritons in crystallization kits, with different different numbers in their names. what's the differences? where can i find their structures? and what does the number mean? From sudhee26 from gmail.com Sun Mar 16 16:04:54 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Mon Mar 17 12:00:25 2008 Subject: "freeze and squeeze" DNA extraction In-Reply-To: References: Message-ID: I use this method.QIAEX II particle. http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000202 try. sudhee. On Sun, Mar 16, 2008 at 3:19 PM, Dunowska, Magda wrote: > Hi, > > Sorry to revisit the subject - I have just gone through the archives and > can't find a description of a method I used to use a few years ago to get > DNA from agarose gels. I used normal (not LMP) agarose and TBE buffer. All I > remember is that it involved freezing and thawing a gel slice, but I think I > was also adding water or TE (can't remember what and how much), heating the > thawed gel to either 65 or 70 degrees for 5 or 10 minutes (?) and mashing it > all up with the blue tip before centrifuging everything at 4 degrees to > collect supernatant. > > > > I just got back to the molecular work after a few years in a slightly > different role, and (surprisingly...) can't recall all the details of > procedures that I used to do on a daily basis and never bothered to write > down outside of the lab books that are no longer with me...Anybody out there > who uses/used a similar variant of the "freeze and squeeze" and can give me > the details? > > Thanks! > > Magda > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From karapini from gmail.com Mon Mar 17 04:41:51 2008 From: karapini from gmail.com (popi Arapini) Date: Mon Mar 17 12:00:41 2008 Subject: DMSO in PCR Message-ID: hI! everybody, I'am using DMSO to standardize my PCR (high GC content) I've read in a protocol that, once DMSO in the thermowell tube, it shall not be spinned down . Does anybody know why?? Popi From poupakfar from yahoo.com Mon Mar 17 12:07:01 2008 From: poupakfar from yahoo.com (Farahani Poupak) Date: Mon Mar 17 13:33:11 2008 Subject: "freeze and squeeze" DNA extraction In-Reply-To: Message-ID: <54812.85425.qm@web51610.mail.re2.yahoo.com> I just use "Freeze 'N Squeeze DNA Gel Extraction Kit" from Bio-Rad. I don't have any variants for this protocol. --- Sudheendra Rao N R wrote: > I use this method.QIAEX II particle. > http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000202 > > try. > sudhee. > > On Sun, Mar 16, 2008 at 3:19 PM, Dunowska, Magda > > wrote: > > > Hi, > > > > Sorry to revisit the subject - I have just gone > through the archives and > > can't find a description of a method I used to use > a few years ago to get > > DNA from agarose gels. I used normal (not LMP) > agarose and TBE buffer. All I > > remember is that it involved freezing and thawing > a gel slice, but I think I > > was also adding water or TE (can't remember what > and how much), heating the > > thawed gel to either 65 or 70 degrees for 5 or 10 > minutes (?) and mashing it > > all up with the blue tip before centrifuging > everything at 4 degrees to > > collect supernatant. > > > > > > > > I just got back to the molecular work after a few > years in a slightly > > different role, and (surprisingly...) can't recall > all the details of > > procedures that I used to do on a daily basis and > never bothered to write > > down outside of the lab books that are no longer > with me...Anybody out there > > who uses/used a similar variant of the "freeze and > squeeze" and can give me > > the details? > > > > Thanks! > > > > Magda > > > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > ____________________________________________________________________________________ Never miss a thing. Make Yahoo your home page. http://www.yahoo.com/r/hs From carla.beukes from gmail.com Tue Mar 18 00:49:49 2008 From: carla.beukes from gmail.com (Carla Beukes) Date: Tue Mar 18 10:06:26 2008 Subject: Digestion Problem Message-ID: Thank you so much. Will certainly try that. Carla From ivanoov from gmail.com Tue Mar 18 05:46:08 2008 From: ivanoov from gmail.com (chovek69) Date: Tue Mar 18 10:06:38 2008 Subject: DMSO in PCR References: <7NCDj.51$XB6.34@newsfe06.lga> Message-ID: On Mar 18, 1:03?am, d...@no.email.thankstospam.net (DK) wrote: > In article , "popi Arapini" wrote: > > >hI! everybody, > >I'am using DMSO to standardize my PCR (high GC content) > >I've read in a protocol that, once DMSO in the thermowell tube, it shall > >not ?be spinned ?down . Does anybody ?know why?? > > No reason. You heard wrong. Yes this is nonsense. 5-10% DMSO disolves perfectly in PCR buffer .... so no bother. In our lab 5% DMSO seems best for many assays. From virashkgupta from gmail.com Tue Mar 18 08:59:08 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Tue Mar 18 12:14:45 2008 Subject: Subject: "freeze and squeeze" DNA extraction Message-ID: What you are doing is OK. In a 0.5 ml tube make a fine pore at the bottom. Cover the hole with some sterile glass wool. Put the mashed slice. keep this tube in 1.5 ml tube ans spin hard in microcentrifuge at maximum speed for 5 min. DNA comes in the outer tube. You can use directly or after precipitation. all the best. On Mon, Mar 17, 2008 at 10:36 PM, wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: Digestion Problem (WS) > 2. "freeze and squeeze" DNA extraction (Dunowska, Magda) > 3. colony PCR method for Bacillus subtilis (Amr Negm) > 4. "Triton"s (kalva) > 5. Re: "freeze and squeeze" DNA extraction (Sudheendra Rao N R) > 6. DMSO in PCR (popi Arapini) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sat, 15 Mar 2008 18:45:00 -0700 (PDT) > From: WS > Subject: Re: Digestion Problem > To: methods@net.bio.net > Message-ID: > <04e4c322-b906-4632-b730-288db2f14131@c33g2000hsd.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi Carla, > > if you get a band with your PCR, you can see the marker on the gel but > there is no signal after the digest, you most likely have a DNAse > somewhere in your system. Be sure to include sufficient and suitable > controls for the digest (in your case, PCR product on the gel should > be enough). To locate the possible source of DNA contamination, you > might want to leave out *one* component of the digest at a time. Maybe > it's more straightforward to get everything new (water, buffer) than > to try around for a long time. > > HTH > > Wo > > > ------------------------------ > > Message: 2 > Date: Sun, 16 Mar 2008 22:49:48 +1300 > From: "Dunowska, Magda" > Subject: "freeze and squeeze" DNA extraction > To: > Message-ID: > > Content-Type: text/plain; charset="utf-8" > > Hi, > > Sorry to revisit the subject - I have just gone through the archives and > can't find a description of a method I used to use a few years ago to get > DNA from agarose gels. I used normal (not LMP) agarose and TBE buffer. All I > remember is that it involved freezing and thawing a gel slice, but I think I > was also adding water or TE (can't remember what and how much), heating the > thawed gel to either 65 or 70 degrees for 5 or 10 minutes (?) and mashing it > all up with the blue tip before centrifuging everything at 4 degrees to > collect supernatant. > > > > I just got back to the molecular work after a few years in a slightly > different role, and (surprisingly...) can't recall all the details of > procedures that I used to do on a daily basis and never bothered to write > down outside of the lab books that are no longer with me...Anybody out there > who uses/used a similar variant of the "freeze and squeeze" and can give me > the details? > > Thanks! > > Magda > > > > > > > ------------------------------ > > Message: 3 > Date: Sat, 15 Mar 2008 17:29:13 -0700 (PDT) > From: Amr Negm > Subject: colony PCR method for Bacillus subtilis > To: methods@oat.bio.indiana.edu > Message-ID: <136023.98989.qm@web55615.mail.re4.yahoo.com> > Content-Type: text/plain; charset=iso-8859-1 > > > I am in need of colony PCR method for > Bacillus subtilis. > I got the positive colonies for but i can > not make this PCR > > waiting for help > > Amr > > > > --------------------------------- > Looking for last minute shopping deals? Find them fast with Yahoo! > Search. > > ------------------------------ > > Message: 4 > Date: Sun, 16 Mar 2008 05:35:02 -0700 (PDT) > From: kalva > Subject: "Triton"s > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > when extract proteins from membrane, usually we try Triton X100 first, > but there are different kinds of Tritons in crystallization kits, with > different different numbers in their names. what's the differences? > where can i find their structures? and what does the number mean? > > > ------------------------------ > > Message: 5 > Date: Mon, 17 Mar 2008 02:34:54 +0530 > From: "Sudheendra Rao N R" > Subject: Re: "freeze and squeeze" DNA extraction > To: "Dunowska, Magda" , methods > > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > I use this method.QIAEX II particle. > http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000202 > > try. > sudhee. > > On Sun, Mar 16, 2008 at 3:19 PM, Dunowska, Magda > wrote: > > > Hi, > > > > Sorry to revisit the subject - I have just gone through the archives and > > can't find a description of a method I used to use a few years ago to > get > > DNA from agarose gels. I used normal (not LMP) agarose and TBE buffer. > All I > > remember is that it involved freezing and thawing a gel slice, but I > think I > > was also adding water or TE (can't remember what and how much), heating > the > > thawed gel to either 65 or 70 degrees for 5 or 10 minutes (?) and > mashing it > > all up with the blue tip before centrifuging everything at 4 degrees to > > collect supernatant. > > > > > > > > I just got back to the molecular work after a few years in a slightly > > different role, and (surprisingly...) can't recall all the details of > > procedures that I used to do on a daily basis and never bothered to > write > > down outside of the lab books that are no longer with me...Anybody out > there > > who uses/used a similar variant of the "freeze and squeeze" and can give > me > > the details? > > > > Thanks! > > > > Magda > > > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > Message: 6 > Date: Mon, 17 Mar 2008 11:41:51 +0200 > From: "popi Arapini" > Subject: DMSO in PCR > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > hI! everybody, > I'am using DMSO to standardize my PCR (high GC content) > I've read in a protocol that, once DMSO in the thermowell tube, it shall > not be spinned down . Does anybody know why?? > > Popi > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 34, Issue 17 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From sidharth4 from gmail.com Wed Mar 19 05:59:26 2008 From: sidharth4 from gmail.com (Siddhartha Parida) Date: Wed Mar 19 11:44:58 2008 Subject: Help Message-ID: What is composition of electro transfer buffer for transferring DNA to nitrocellulose membranes by electroblotting ? On Tue, Mar 18, 2008 at 10:34 PM, wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: "freeze and squeeze" DNA extraction (Farahani Poupak) > 2. Re: Digestion Problem (Carla Beukes) > 3. Re: DMSO in PCR (DK) > 4. Re: DMSO in PCR (chovek69) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 17 Mar 2008 10:07:01 -0700 (PDT) > From: Farahani Poupak > Subject: Re: "freeze and squeeze" DNA extraction > To: Sudheendra Rao N R , "Dunowska, Magda" > , methods > Message-ID: <54812.85425.qm@web51610.mail.re2.yahoo.com> > Content-Type: text/plain; charset=iso-8859-1 > > I just use "Freeze 'N Squeeze DNA Gel Extraction Kit" > from Bio-Rad. I don't have any variants for this > protocol. > > > > > --- Sudheendra Rao N R wrote: > > > I use this method.QIAEX II particle. > > > http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000202 > > > > try. > > sudhee. > > > > On Sun, Mar 16, 2008 at 3:19 PM, Dunowska, Magda > > > > wrote: > > > > > Hi, > > > > > > Sorry to revisit the subject - I have just gone > > through the archives and > > > can't find a description of a method I used to use > > a few years ago to get > > > DNA from agarose gels. I used normal (not LMP) > > agarose and TBE buffer. All I > > > remember is that it involved freezing and thawing > > a gel slice, but I think I > > > was also adding water or TE (can't remember what > > and how much), heating the > > > thawed gel to either 65 or 70 degrees for 5 or 10 > > minutes (?) and mashing it > > > all up with the blue tip before centrifuging > > everything at 4 degrees to > > > collect supernatant. > > > > > > > > > > > > I just got back to the molecular work after a few > > years in a slightly > > > different role, and (surprisingly...) can't recall > > all the details of > > > procedures that I used to do on a daily basis and > > never bothered to write > > > down outside of the lab books that are no longer > > with me...Anybody out there > > > who uses/used a similar variant of the "freeze and > > squeeze" and can give me > > > the details? > > > > > > Thanks! > > > > > > Magda > > > > > > > > > > > > > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > > > -- > > Think before agree > > Think before you nod > > but STOP thinking > > and You Are God > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > ____________________________________________________________________________________ > Never miss a thing. Make Yahoo your home page. > http://www.yahoo.com/r/hs > > > > ------------------------------ > > Message: 2 > Date: Tue, 18 Mar 2008 07:49:49 +0200 > From: "Carla Beukes" > Subject: Re: Digestion Problem > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Thank you so much. Will certainly try that. > > Carla > > > ------------------------------ > > Message: 3 > Date: Mon, 17 Mar 2008 23:03:30 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: DMSO in PCR > To: methods@net.bio.net > Message-ID: <7NCDj.51$XB6.34@newsfe06.lga> > > In article , "popi Arapini" wrote: > >hI! everybody, > >I'am using DMSO to standardize my PCR (high GC content) > >I've read in a protocol that, once DMSO in the thermowell tube, it shall > >not be spinned down . Does anybody know why?? > > No reason. You heard wrong. > > > > ------------------------------ > > Message: 4 > Date: Tue, 18 Mar 2008 03:46:08 -0700 (PDT) > From: chovek69 > Subject: Re: DMSO in PCR > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > On Mar 18, 1:03 am, d...@no.email.thankstospam.net (DK) wrote: > > In article , "popi Arapini" wrote: > > > > >hI! everybody, > > >I'am using DMSO to standardize my PCR (high GC content) > > >I've read in a protocol that, once DMSO in the thermowell tube, it shall > > >not be spinned down . Does anybody know why?? > > > > No reason. You heard wrong. > > Yes this is nonsense. 5-10% DMSO disolves perfectly in PCR buffer .... > so no bother. In our lab 5% DMSO seems best for many assays. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 34, Issue 18 > *************************************** > -- Sidharth Parida Senior Faculty Center for biotechnology and Research Neelachal Inst of Medical Sciences O.C.H.C Complex, Block - III (Near Ram Mandir) Janpath,Unit-3,Bhubaneswar- 751001 ORISSA 09437089337(M) sidharth4@gmail.com www.nimsindia.com From sudhee26 from gmail.com Wed Mar 19 23:08:48 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Mar 20 13:41:57 2008 Subject: Help In-Reply-To: References: Message-ID: I use the following transfer buffer. for 500ml 350ml MilliQ Water 50ml 10X Electrode Buffer (Tris.Glycine-pH 8.3) 100ml Methanol Per blot 50-80ml is sufficient (semi Dry transfer). I dont re-use the transfer buffer..instead i keep used ones to take out ponceau staining after its done. Sudheendra. On Wed, Mar 19, 2008 at 4:29 PM, Siddhartha Parida wrote: > What is composition of electro transfer buffer for transferring DNA to > nitrocellulose membranes > by electroblotting ? > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God From engelbert_buxbaum from hotmail.com Fri Mar 21 12:42:10 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Sat Mar 22 06:07:22 2008 Subject: "Triton"s References: Message-ID: Am 16.03.2008, 08:35 Uhr, schrieb kalva : > when extract proteins from membrane, usually we try Triton X100 first, > but there are different kinds of Tritons in crystallization kits, with > different different numbers in their names. what's the differences? > where can i find their structures? and what does the number mean? The average length of the ethylenglycol chain is different, resulting in different properties (cmc, \bar{m}, Kraft point). Remember that Tritons are industrial detergents, that is wild mixtures of related compounds. For crystallisation I'd prefer detergents with defined structure, e.g. C12E8 or the like. Some detergent properties you can get from http://psyche.uthct.edu/shaun/SBlack/detergnt.html, also from the web pages of manufacturers like Anatrace. From cbejar from berkeley.edu Thu Mar 20 13:47:54 2008 From: cbejar from berkeley.edu (Clarisa Bejar) Date: Sat Mar 22 06:19:38 2008 Subject: Annealing temp using Quikchange SDM kit Message-ID: Hi, I?m designing my Quikchange-SDM and have a question regarding the annealing temperature. What is the best way to determine the annealing temperature since, in this case, primers should be designed with a minimum Tm= 78?C? (so the annealing temperature= Tm- 5?C wouldn?t apply here) Thanks for your feedback! Clarisa From wshen4 from jhmi.edu Fri Mar 21 13:26:24 2008 From: wshen4 from jhmi.edu (jhmi) Date: Sat Mar 22 06:19:54 2008 Subject: Umea Drosophila stocks Message-ID: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> SGksIERyIERvbiBHaWxiZXJ0LA0KDQpJJ20gYSBzdHVkZW50IGZyb20gSm9obnMgSG9wa2lucy4g Q291bGQgeW91IGRvIG1lIGEgZmF2b3IgdG8gdGVsbCBtZSBob3cgdG8gYWNjZXNzIFVtZWEgU3Rv Y2sgQ2VudHJlLiBJIHdhbnQgdG8gb3JkZXIgc29tZSBmbHkgc3RvY2sgZnJvbSBpdC4gDQpUaGFu a3MgYSBsb3QuDQoNCldlaSBTaGVuDQpCaW9sb2dpY2FsIENoZW1pc3RyeQ0KSkhNSQ== From tk from mit.edu Sat Mar 22 06:43:09 2008 From: tk from mit.edu (Tom Knight) Date: Sat Mar 22 07:40:28 2008 Subject: Annealing temp using Quikchange SDM kit References: Message-ID: "Clarisa Bejar" writes: > I'm designing my Quikchange-SDM and have a question regarding the > annealing temperature. What is the best way to determine the annealing > temperature since, in this case, primers should be designed with a > minimum Tm= 78?C? (so the annealing temperature= Tm- 5?C wouldn't > apply here) For Quikchange, both the 3' and 5' ends of the primer must bind well. The easiest way to assure this is to pretend you are designing two different primers, one 5' of the mutation, and a second 3' of the mutation, and get the Tm of these primers roughtly correct. The manual tells you what you need to know. The Tm's that any program calculates will just confuse you, and you should ignore them. In general, design 16-20 bp matching, surrounding the mutated region, more if there is high GC content. You might try for high GC at the extreme 5' and 3' ends. From sudhee26 from gmail.com Sun Mar 23 06:17:57 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Mon Mar 24 12:38:41 2008 Subject: Umea Drosophila stocks In-Reply-To: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> References: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> Message-ID: any help? http://bacpac.chori.org/drosofosmid.htm Sudheendra. On Fri, Mar 21, 2008 at 11:56 PM, jhmi wrote: > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how > to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. > > Wei Shen > Biological Chemistry > JHMI > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From matsubayashi from DGRC.kit.ac.jp Sun Mar 23 20:57:24 2008 From: matsubayashi from DGRC.kit.ac.jp (MATSUBAYASHI, Hiroshi) Date: Mon Mar 24 12:38:57 2008 Subject: Umea Drosophila stocks In-Reply-To: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> References: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> Message-ID: <20080324105724.Postino-011027@192.168.0.9> > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. Umea Stock Centre was closed in many years ago, and part of the stocks moved to Drosophila Genetic Resource Center (DGRC) in Kyoto. Please see the website, http://www.dgrc.kit.ac.jp/en/ From sudhee26 from gmail.com Tue Mar 25 00:43:06 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Mar 25 11:33:45 2008 Subject: Fwd: Umea Drosophila stocks In-Reply-To: <20080324105724.Postino-011027@192.168.0.9> References: <000e01c88b81$14e4f350$5b4a100a@71e0c427bf73479> <20080324105724.Postino-011027@192.168.0.9> Message-ID: ---------- Forwarded message ---------- From: MATSUBAYASHI, Hiroshi Date: Mon, Mar 24, 2008 at 7:27 AM Subject: Re: Umea Drosophila stocks To: methods@magpie.bio.indiana.edu > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. Umea Stock Centre was closed in many years ago, and part of the stocks moved to Drosophila Genetic Resource Center (DGRC) in Kyoto. Please see the website, http://www.dgrc.kit.ac.jp/en/ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods -- Think before agree Think before you nod but STOP thinking and You Are God From Ajay.Niranjane from csiro.au Tue Mar 25 17:33:21 2008 From: Ajay.Niranjane from csiro.au (Ajay.Niranjane@csiro.au) Date: Wed Mar 26 12:01:03 2008 Subject: Methods Digest, Vol 34, Issue 23 References: <200803251705.m2PH5EL08228@net.bio.net> Message-ID: <5EC14D1CCAE30041811932B07552011B01307E31@exactn1-cbr.nexus.csiro.au> Hi All, I am trying to express a highly repetitive protein in E.coli using Rosetta gami expression cell line. Can anyone explain the role of chloramphenicol in expression of rare codons? Thanks in advance. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Wednesday, 26 March 2008 4:05 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 34, Issue 23 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-request@net.bio.net You can reach the person managing the list at methods-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. Re: Umea Drosophila stocks (MATSUBAYASHI, Hiroshi) 2. Re: Umea Drosophila stocks (Sudheendra Rao N R) 3. Fwd: Umea Drosophila stocks (Sudheendra Rao N R) ---------------------------------------------------------------------- Message: 1 Date: Mon, 24 Mar 2008 10:57:24 +0900 From: "MATSUBAYASHI, Hiroshi" Subject: Re: Umea Drosophila stocks To: methods@magpie.bio.indiana.edu Message-ID: <20080324105724.Postino-011027@192.168.0.9> Content-Type: text/plain; charset=us-ascii > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. Umea Stock Centre was closed in many years ago, and part of the stocks moved to Drosophila Genetic Resource Center (DGRC) in Kyoto. Please see the website, http://www.dgrc.kit.ac.jp/en/ ------------------------------ Message: 2 Date: Sun, 23 Mar 2008 16:47:57 +0530 From: "Sudheendra Rao N R" Subject: Re: Umea Drosophila stocks To: jhmi , methods Message-ID: Content-Type: text/plain; charset=ISO-8859-1 any help? http://bacpac.chori.org/drosofosmid.htm Sudheendra. On Fri, Mar 21, 2008 at 11:56 PM, jhmi wrote: > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how > to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. > > Wei Shen > Biological Chemistry > JHMI > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God ------------------------------ Message: 3 Date: Tue, 25 Mar 2008 11:13:06 +0530 From: "Sudheendra Rao N R" Subject: Fwd: Umea Drosophila stocks To: jhmi , methods Message-ID: Content-Type: text/plain; charset=ISO-8859-1 ---------- Forwarded message ---------- From: MATSUBAYASHI, Hiroshi Date: Mon, Mar 24, 2008 at 7:27 AM Subject: Re: Umea Drosophila stocks To: methods@magpie.bio.indiana.edu > Hi, Dr Don Gilbert, > > I'm a student from Johns Hopkins. Could you do me a favor to tell me how to access Umea Stock Centre. I want to order some fly stock from it. > Thanks a lot. Umea Stock Centre was closed in many years ago, and part of the stocks moved to Drosophila Genetic Resource Center (DGRC) in Kyoto. Please see the website, http://www.dgrc.kit.ac.jp/en/ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods -- Think before agree Think before you nod but STOP thinking and You Are God ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 34, Issue 23 *************************************** From aswinisekar16 from gmail.com Tue Mar 25 18:16:18 2008 From: aswinisekar16 from gmail.com (aswini sekar) Date: Wed Mar 26 12:01:12 2008 Subject: S1 nuclease on DNA protocol Message-ID: <95ccb3c10803251616w4a9cf892xe7b5cdd90c10e35f@mail.gmail.com> Dear friends, I am aswini doing my project in molecular biology. I need a short protocol for the treatment of S1 nuclease on DNA . Its very urgent.Pl... do help me. Thank u Aswini From wxfhome from gmail.com Wed Mar 26 06:14:27 2008 From: wxfhome from gmail.com (WANG XF) Date: Wed Mar 26 12:01:19 2008 Subject: How to express a protein (small peptide) as small as 5kd? Message-ID: <404daa2f0803260414v4a4023d6q86f7d37560f14ea8@mail.gmail.com> How to express a protein (small peptide) as small as 5kd? In E.coli or mammalian cell? Thank you all. WANG From blackhole from abuse.plus.com Thu Mar 27 05:57:37 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Mar 27 13:24:15 2008 Subject: Methods Digest, Vol 34, Issue 23 References: <200803251705.m2PH5EL08228@net.bio.net> Message-ID: Historians believe that in newspost on Wed, 26 Mar 2008, Ajay.Niranjane@csiro.au penned the following literary masterpiece: >Can anyone explain the role of chloramphenicol in expression of rare >codons? >Thanks in advance. Well, how are the extra tRNA's for those rare codons supplied in the Rosetta gami strain? Having found that out from the strain literature you should be able to see why CAT is used. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From sudhee26 from gmail.com Wed Mar 26 22:52:35 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Mar 27 13:24:25 2008 Subject: S1 nuclease on DNA protocol In-Reply-To: <95ccb3c10803251616w4a9cf892xe7b5cdd90c10e35f@mail.gmail.com> References: <95ccb3c10803251616w4a9cf892xe7b5cdd90c10e35f@mail.gmail.com> Message-ID: http://www.fermentas.com/techinfo/modifyingenzymes/protocols/p_generunidirdeldna.htm I guess, getting DNA after restriction digestion is going to take little time..may be someone else will give you that protocol..or u can google for it.Rest i guess is workable. All the best Sudheendra NBRC, Gurgaon. On Wed, Mar 26, 2008 at 4:46 AM, aswini sekar wrote: > Dear friends, > I am aswini doing my project in molecular biology. > I need a short protocol for the treatment of S1 nuclease on DNA . > Its very urgent.Pl... do help me. > Thank u > Aswini > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Wed Mar 26 22:54:01 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Mar 27 13:24:31 2008 Subject: S1 nuclease on DNA protocol In-Reply-To: References: <95ccb3c10803251616w4a9cf892xe7b5cdd90c10e35f@mail.gmail.com> Message-ID: You can get a better protocol if you state the application u are willing to use S1 Nuclease in. Regards Sudheendra. On Thu, Mar 27, 2008 at 9:22 AM, Sudheendra Rao N R wrote: > > http://www.fermentas.com/techinfo/modifyingenzymes/protocols/p_generunidirdeldna.htm > > I guess, getting DNA after restriction digestion is going to take little > time..may be someone else will give you that protocol..or u can google for > it.Rest i guess is workable. > > All the best > > Sudheendra > NBRC, Gurgaon. > > On Wed, Mar 26, 2008 at 4:46 AM, aswini sekar > wrote: > > > Dear friends, > > I am aswini doing my project in molecular biology. > > I need a short protocol for the treatment of S1 nuclease on DNA > > . > > Its very urgent.Pl... do help me. > > Thank u > > Aswini > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God -- Think before agree Think before you nod but STOP thinking and You Are God From cv2135 from columbia.edu Thu Mar 27 17:30:09 2008 From: cv2135 from columbia.edu (cv2135@columbia.edu) Date: Thu Mar 27 18:52:24 2008 Subject: Transformation onto M9 Minimal Media Message-ID: <20080327183009.4hzghjnstcokwks8@cubmail.cc.columbia.edu> Hello I have a toxic protein expressed from a T7 promoter in Pet21 D. I'm trying to harvest the protein, but getting little yield. I was told to do everything in minimal media to reduce any leaky expression of T7 (I.e. to make certain there is no break down of lactose analogs found in LB). I am using M8 MM + .4% glucose + 1mg/ML CAA + 100 ug/ML Ampicillin Unfortunately, I am having a hard time even TRANSFORMING my plasmids into BL21(DE3)pLysS (there should be little expression in this strain anyway) in Minimal Media. Even the puc 19 positive control failed. I did the typical 30 minutes on ice, 45 sec heat shock at 42C, 2 minutes ice, 1 hour recovery (in M9 MM) . twice this has failed (it also failed once on E.coli). IS there any reason I should have such problems? Please help if you can thanks Christal From nirosha.mab from ualberta.ca Thu Mar 27 16:16:44 2008 From: nirosha.mab from ualberta.ca (Nirosha Gunasekara) Date: Thu Mar 27 18:52:38 2008 Subject: Need help to find a protein expression protocol for cysteine cross linked dimers Message-ID: <20080327151644.zu9me2bxyoo08sw4@webmail.ualberta.ca> Hello All, I need help to choose a vector to express a cysteine cross linked dimeric fragment and the monomeric fragment of a protien for NMR structure determination. Since I need to express just a part of a protein what are the limitations (or things to consider) when choosing an expression vector as well as purification of dimeric and monomeric proteins? Which vector(s) may be good candidates? Does choosing a vector depend on the cysteine cross linking procedure? I am wondering whether vector, pEZZ18 (Amersham Bio) can be used to express just a part of a protien as I read that the expression by pEZZ18 is controlled by the lacUV5 and protein A promoters and is not inducible. I am new to molecular cloning and could you please clarify this as well. If you could suggest a common protocol for the expression of cysteine cross linked dimers or provide me with a direction to look for that would be ideal. All your help is greatly appreciated. Thank you very much! Nirosha From scoop from mail.nih.gov Thu Mar 27 22:36:03 2008 From: scoop from mail.nih.gov (Sharon) Date: Fri Mar 28 11:34:25 2008 Subject: migration of dyes and DNA thru alkaline agarose gels Message-ID: Dear Experts, I need to run an alkaline agarose gel to crudely measure the length of a primer extension reaction (prior to doing a urea gel to closely and accurately measure length). Does anyone know what the migration of bromophenol blue and xylene cyanol are in alkaline agarose gels relative to DNA size (or alkaline polyacrylamide gels)? Thanks, Sharon From Paul.Phelan from tufts.edu Thu Mar 27 18:45:28 2008 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Fri Mar 28 11:34:56 2008 Subject: GST contamination of GST fusion protein Message-ID: <20080327194528.h5hd7rvdc8ssk0ow@webmail.tufts.edu> To whom it concerns, This question maybe have been asked before, but I am having trouble with separating GST from a preparation of GST-Importin alpha. I have maybe 10-20% GST co-purifying with the fusion protein. The pI of both proteins is the same, so ion exchange chromatography does not separate them, and even on a Sephacryl S-100 gel filtration column, the two proteins (87 kDa fusion, 27 kDa GST) eluted together. Does anyone have a clever trick to remove GST from a GST fusion prep.? My next attempt is going to be to try gel filtration again but with 0.3 M NaCl and maybe some detergent to try to separate the proteins. Any advice would be greatly appreciated, thanks Paul Phelan Tufts University Biochemistry Dept. Boston From aawara from pontiff-playground.org Sat Mar 29 07:14:06 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat Mar 29 12:57:02 2008 Subject: GST contamination of GST fusion protein References: Message-ID: In , Paul J. Phelan wrote: > To whom it concerns, > This question maybe have been asked before, but I am having trouble > with separating GST from a preparation of GST-Importin alpha. I have > maybe 10-20% GST co-purifying with the fusion protein. The pI of both > proteins is the same, so ion exchange chromatography does not separate > them, and even on a Sephacryl S-100 gel filtration column, the two > proteins (87 kDa fusion, 27 kDa GST) eluted together. Does anyone have > a clever trick to remove GST from a GST fusion prep.? > My next attempt is going to be to try gel filtration again but with 0.3 > M NaCl and maybe some detergent to try to separate the proteins. > > Any advice would be greatly appreciated, thanks GST dimerizes, so it isn't surprising that the cleaved GST moiety co-purifies with your GST-Rch1 fusion. In my limited experience with GST fusions, I've never encountered this premature cleavage that you're seeing, so my suggestion below is not tested. Why don't you denature your GST/GST-Rch1 mixture, and then separate them by gel filtration? Using 3M GdHCl? AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From Antonio.Sarikas from gmail.com Mon Mar 31 18:03:03 2008 From: Antonio.Sarikas from gmail.com (AS) Date: Mon Mar 31 18:51:37 2008 Subject: soft agar transformation assay Message-ID: Hi, could anyone recommend a protocol for a soft agar transfromation assays for mouse embryonic fibroblasts and human diploid fibroblasts? Some technical questions: 1. which agarose do you use (DNA grade?) 2. how do you get the agarase sterile? 3. which medium do you use: 2x DMEM? Your advice is greatly appreciated - thanks! AS