most recent 30 from http://www.majorgroove.org 2010-09-09T19:17:08Z http://www.majorgroove.org/feeds http://www.creativecommons.org/licenses/by-nc/2.5/rdf http://www.majorgroove.org/questions/276/nested-pcr-rt-pcr Rosh 2010-06-29T15:52:48Z 2010-09-09T14:24:12Z

<p>Hi, I am just wandering if it is possible to do nested PCR with the same primer used primarily? would appreciate suggestion on this matter.</p>

http://www.majorgroove.org/questions/83/refmac-5-5-0092-not-writing-pdb-mtz-files Ben Eisenbraun 2010-01-29T04:59:31Z 2010-09-09T01:24:09Z

<p>Howdy,</p> <p>One of our users was trying to re-run some Refmac jobs originally run with version 5.5.0088 under version 5.5.0092, and he noticed that although the logfile shows Refmac completing successfully, the new version is not actually writing the pdb/mtz output files.</p> <p>He is using linux with CCP4 6.1.1 as the interface with the latest patches from the problems page. Is this a known issue with Refmac? I notice that Garib has put up a few more source releases on his personal page, but there wasn't an update to the "bug removal and new features" page, so I don't know if the new versions address this problem.</p> <p>The new Refmac binaries for OS X Intel only run on 10.5. It would be nice if the build scripts set the environment for:</p> <blockquote> <p>MACOSX_DEPLOYMENT_TARGET=10.4</p> </blockquote> <p>So the binaries would run on Tiger. (Maybe need a -mmacosx-version-min=10.4 in the CFLAGS/LDFLAGS as well.)</p> <p>-ben</p> <p>Ben Eisenbraun | Software Maintainer<br /> Structural Biology Grid | <a href="http://sbgrid.org" rel="nofollow">http://sbgrid.org</a><br /> Harvard Medical School </p>

http://www.majorgroove.org/questions/42/recommendation-for-dynamic-static-light-scattering-unit Alexandra Deaconescu 2010-01-25T02:22:28Z 2010-09-08T23:24:10Z

<p>We are thinking of buying a dynamic/static light scattering unit to analyze our samples. The <a href="http://www.wyatt.eu/index.php?id=dynapro%5Fnanostar" rel="nofollow">Nanostar from Wyatt</a> (previously from Dynapro) seems to combine both DLS and static light scattering. I was wondering if anyone has experience with it? How good/accurate are the static light scattering measurements compared to the 18-angle Heleos or 3-angle Treos? It looks like the instrument is primarily designed for DLS, so I was wondering how bad the static scattering detector is. We are primarily interested in getting an estimation for absolute MWs, and we would like to have temperature control, which the Heleos seems to lack...</p> <p>Thank you so much.</p> <p>Alex</p>

http://www.majorgroove.org/questions/296/jobs-for-biologists Paul Lindblom 2010-08-24T12:38:26Z 2010-09-07T19:24:09Z

<p>Dear scientists,</p> <p>at the moment I am writing up my PhD thesis. In parallel I am looking for a new job outside the lab. I had a lot of fun during my PhD and it was a great and interesting time, but I decided for my future to do no postdoc, but something different where I can start a family and can share some time.</p> <p>So my question is: Does anyone have suggestions what a (structural) biologist can do outside the lab? Does anyone gained experience? I just study vaccancies in the field of "drug regulatory affairs" and it seems that there are a lot of open positions...</p> <p>Thanks a lot in advance,</p> <p>Paul. </p>

http://www.majorgroove.org/questions/303/i-am-trying-to-run-a-plant-extract-in-a-native-page-but-the-sample-doesnt-run-i Mir 2010-09-02T17:02:34Z 2010-09-03T18:10:37Z

<p>I am trying to run a plant extract in a native PAGE but the sample doesn't run, if run a little bit it gives a protein smear. Does anybody knows how can I solve the problem?</p>

http://www.majorgroove.org/questions/257/which-hrp-grade-for-antibody-labeling WS 2010-04-30T10:49:39Z 2010-09-03T13:24:15Z

<p>Dear Experts,</p> <p>I would like to label an antibody with peroxidase by the periodate method. Which HRP grade / isoforms / purity should I use? Is there a practical advantage (or even disadvantage) of "stabilized" peroxidase, where the amino groups are chemically protected and therefore oligomerization of the HRP would be prevented?</p> <p>The AB will be used in westerns and Elisa.</p> <p>Many thanks!</p> <p>Wo </p>

http://www.majorgroove.org/questions/192/list-of-biology-blogs Simon Tustin 2010-03-05T19:49:17Z 2010-09-02T20:24:16Z

<p>I thought it might be valuable to have a list of blogs that people find useful. If you're a registered user on MajorGroove, feel free to update this list directly by clicking <em>edit</em> under the list of tags. This post is a <em>community wiki</em>.</p> <ul> <li><a href="http://bitesizebio.com/" rel="nofollow">Bitesize Bio</a> - Cell and molecular biology</li> <li><a href="http://www.p212121.com/" rel="nofollow">P212121.com</a> - Crystallography</li> <li><a href="http://web.emeraldbiosystems.com/" rel="nofollow">Protein Crystallization Hits</a> - Crystallisation</li> <li><a href="http://blogs.nature.com/scurry/" rel="nofollow">Reciprocal Space</a> - by Stephen Curry - science, opinion and crystallography (good stuff)</li> <li><a href="http://pipeline.corante.com/" rel="nofollow">In the Pipeline</a> - by Derek Lowe - biotech (probably will know that your company is laying you off here first), med chem and chemistry stories about how to kill yourself</li> <li><a href="http://phylogenomics.blogspot.com/" rel="nofollow">Tree of Life</a> - by Jonathan A. Eisen (Evolution)</li> </ul>

http://www.majorgroove.org/questions/304/how-can-i-remove-a-myc-tag-from-my-overexpressed-protein Mir 2010-09-02T17:04:18Z 2010-09-02T19:29:13Z

<p>how can I remove a myc-tag from my overexpressed protein?</p>

http://www.majorgroove.org/questions/50/is-modip-still-available-any-alternatives Mark Del Campo 2010-01-25T06:16:27Z 2010-09-02T16:29:37Z

<p>Does anyone know if the program MODIP is still available? Anyone know any other programs that will do what MODIP does?</p> <p>Thanks!</p> <p>Mark</p>

http://www.majorgroove.org/questions/295/which-polymerase-is-most-suited-for-incorporating-exotic-cy-labeled-ntps unknown (google) 2010-08-19T12:02:21Z 2010-09-02T13:24:24Z

<p>Well, obviously choosing a suitable polymerase for second strand ligation depends on a whole lot of experimental parameters. In my case the most crucial parameter is the efficient incorporation of a cyanine-3 labeled nucleotide at the end of the template, which is just about 60nt long. Unfortunately, I may have to chose a more exotic nucleotide such as Cy-3 dGTP and quite frankly there is next to no information out there about purine-analogues that have a dye attached and the efficiency of any polymerase to incorporate such analogs during strand extension. </p> <p>I am considering using Taq-polymerase which seems to be fairly tolerant but that just get me into the next living hell of picking one Taq-variant from the various suppliers. </p> <p>I was wondering if anybody has any experience regarding this setup and can recommend or dis-advise the use of a specific polymerase. </p>

http://www.majorgroove.org/questions/30/gpu-computing-in-structural-biology Simon Tustin 2010-01-23T22:12:42Z 2010-09-01T18:24:05Z

<p>I'm aware of GPU computing making <a href="http://www.bit-tech.net/news/bits/2009/12/15/astra-builds-13-gpu-supercomputer/1" rel="nofollow">great strides in tomography</a>, but are there any projects which use GPGPUs in structural biology? Or, are the algorithms from, for example, crystallography not amenable to use on GPUs?</p>

http://www.majorgroove.org/questions/250/lysine-methylation-for-proteins-containing-disulfide-bonds Oliver Clarke 2010-04-14T01:29:32Z 2010-09-01T14:24:06Z

<p>Hi all,</p> <p>I am currently attempting to crystallise a two domain protein which contains several structurally important disulfides. We have a high resolution structure of one domain (1.4 A), which reveals quite a few solvent-exposed lysines, some of which are involved in crystal-contacts.</p> <p>The two-domain construct also crystallises, but the only crystals obtained after extensive optimisation are stacks of thin-plates that show poor diffraction (multiple lattices, streaky spots) to around 3 A.</p> <p>I would like to attempt modification of the lysine residues by reductive methylation or cyclic pentylation, but I am worried that the reducing conditions required for the reaction (due to the presence of the dimethylaminoborane complex) will disrupt the native disulfides and result in protein aggregation or denaturation. Does anyone have any experience with reductive methylation of disulfided proteins, or know of any references describing the same?</p> <p>Thanks in advance, Oliver Clarke.</p>

http://www.majorgroove.org/questions/160/source-for-phe-auxotrophic-e-coli-bl21de3 jflood 2010-02-25T16:57:28Z 2010-08-27T14:24:09Z

<p>Hello, I'm looking for a Phe auxotrophic strain of E Coli that can be used with plasmids with T7 promoter. Preferably BL21 (DE3). Can anybody direct me to a bank, lab or where to buy it? Thnxs.</p>

http://www.majorgroove.org/questions/253/phenomenex-biosep-column-compatibility-with-ge-healthcare-kta-chromatography-sys Roger Dodd 2010-04-22T11:09:27Z 2010-08-27T13:24:05Z

<p>Our lab has an Äkta-based chromatography system and we are looking into using a silica-based gel filtration column. Specifically, we are hoping to use one of the BioSep range of columns from Phenomenex, detailed <a href="http://www.phenomenex.com/cms400min/biosepseccolumns.aspx" rel="nofollow">here</a>.</p> <p>Does anyone have any experience with these columns and would be able to answer: 1. If these columns are compatible with Äkta systems? 2. If any adapters are required? and 3. If there are any other silica-based gel filtration columns to consider?</p>

http://www.majorgroove.org/questions/193/antibody-sticks-to-antigen-affinity-column WS 2010-03-05T22:40:17Z 2010-08-27T12:24:02Z

<p>Dear experts,</p> <p>I try to purify a chicken IgY by affinity chromatography. The hapten (a small "chemical" molecule, not a protein or peptide) is covalently coupled to sepharose.</p> <p>In order to find appropriate conditions, I applied 1mg of IgY (diluted in 100mM tris, pH 7.5) to the column and almost no protein ran through. I tried elution with 2M MgCl2, 5M urea and 100mM gylcine, pH 2.5 successively, washing with 100mM Tris between the steps. I got some small peaks by monitoring the eluate for UV absorption, but the fractions do not contain much protein, so I conclude that the majority of the desired antibody still sticks to the column. Maybe it's of importance that the column has been designed for bulk purification, the capacity is expected to be about 100mg in respect to IgY, so the load for my tests is very low. </p> <p>Any ideas what else I could try? The eluted IgY should be functional, of course. </p> <p>Many thanks for your help!</p> <p>Wo</p>

http://www.majorgroove.org/questions/178/using-windows-pcs-for-crystallography Simon Tustin 2010-03-01T06:24:09Z 2010-08-26T16:27:11Z

<p>I recently asked about <a href="http://www.majorgroove.org/questions/18/are-linux-workstations-still-necessary-for-crystallography-or-will-macs-suffice" rel="nofollow">the state of Macs in crystallography</a>, and now I thought I'd ask about the state of Windows PCs. Windows 7 looks to be pretty good. I know that CCP4, Coot, and PyMol all run under Windows. It appears that HKL2000 does not though. Can anyone fill in the gaps about what runs, and what doesn't?</p> <p>Cheers,</p> <p>Simon </p>

http://www.majorgroove.org/questions/298/the-expression-of-negative-control-is-always-higher-than-that-of-positive-control garry2010 2010-08-26T08:03:21Z 2010-08-26T09:53:20Z

<p>hello everyone!</p> <p>A question about PCR puzzled me many days! I transfected several kinds of siRNA to my tumor cell with lipofectamine. and then testify the transfection result with RT-PCR. But the problem is, the expression of negative control is always higher than that of positive control.</p> <p>I repeated many times,but the result was always similar. i really need your help to analyze and give me some suggestion.</p> <p>THANS A MILLION!!!</p>

http://www.majorgroove.org/questions/293/wanted-receipe-for-a-tmb-based-precipitating-hrp-substrate WS 2010-08-03T07:37:47Z 2010-08-03T07:37:47Z

<p>Hi folks, </p> <p>anyone has a good (of course :) receipe of a TMB based precipitating HRP substrate for westerns? Something like Leinco's HRPO (T116).</p> <p>Thanks for your help!</p> <p>Wo</p>

http://www.majorgroove.org/questions/291/how-long-do-i-have-to-incubate-my-sample-with-trypsin Paul Lindblom 2010-07-27T19:22:25Z 2010-07-28T11:39:01Z

<p>Dear all,</p> <p>does anyone has experience of how long I have to incubate a protein sample with trypsin for a complete digestion? I incubated o.n. at 37°C. Is this generally enough time (independent on trypsin concentration)? And does anyone know if trypsin needs on the left and on the right of the cleavage site some amino acids to work well? Especially for the case of two or more successive cleavage sites?</p> <p>Thank you all very much.</p> <p>Paul</p>

http://www.majorgroove.org/questions/289/in-vitro-gst-pull-down pankaj 2010-07-27T11:24:01Z 2010-07-27T18:01:20Z

<p>hello all, </p> <p>I am doing GST pull down experiment, where my protein tagged with N-terminal GST. and its purified and desalted.</p> <p>when i am doing interaction using Cell lysate (Hela or 293T) containing my pray protein, and after interaction,while doing immunoprobing (Mab) for pray protein i am getting non-specific bands for GST fused protein also, very prominent,</p> <p>i am preclearing cell lysate before interaction. i used differnt salt and buffer compositions, but still the problem persist.</p> <p>i even precleared my antibody also, but then also it didnt work.......</p> <p>plz gimme some suggestions.</p> <p>thank you</p> <p>pankaj</p>

http://www.majorgroove.org/questions/284/ferritin-in-insect-cell-sytem JMB 2010-07-08T07:40:36Z 2010-07-09T07:22:19Z

<p>Hi I express my recombinant protein, with H5 insect cells, directly in the medium (Express Five). During my IMAC purification from the medium, I get a contaminant most likely Ferritin. I can get rid of most of it during the gel filtration step. However a minority remains. Does anyone know a trick to get rid of this ferritin contaminant or prevent insect cells to secrete it??? Thanks in advance</p> <p>JM </p>

http://www.majorgroove.org/questions/282/freezer-organization-record-keeping-what-systems-do-you-use Nicojo 2010-07-05T07:56:21Z 2010-07-09T06:25:55Z

<p>Hi everyone!</p> <p>I started working in my current lab 5 years ago. I started with one box in the refrigerator and one in the freezer... Easy to keep track of things.</p> <p>But today, I have several dozen in each!!! I've been trying to keep track of everything with an excel file, but it's not very efficient.</p> <p>Also, a recent event in my lab also makes me want to get a good system working: my boss has been going crazy over some serum samples from Africa that nobody could find! It took over a year to find them in all the boxes people have accumulated over the years...</p> <p>How do you keep track of your collection of cells, primers, plamsids, sera etc.? Do you use spreadsheets (Excel/OpenOffice), databases (Access, MySQL, FileMaker Pro), pen and paper or something else?</p> <p>I know of different LIMS (Laboratory Information Management System) options, but they all seem to be overkill and mostly too expensive...</p> <p>Please share your successes as well as your failures ;)</p>

http://www.majorgroove.org/questions/277/barspec-chrom-a-scope-anyone WS 2010-06-29T21:25:27Z 2010-06-29T21:25:27Z

<p>Dear all,</p> <p>I found this HPLC spectral detector in a stock room. It's funny stuff with a rotating grid instead of the 'usual' static prism &amp; diode array, said to be very gentle to the sample as the photon load is small. BarSpec, the Israeli company from Rehovot who developed and marketed it, seems not to exist anymore AFIK and the internet only reveals people who were using it some time ago, no manuals and other information.</p> <p>I really would like to reactivate it, as I also have got the software on 5 1/4" floppies (wonder if they are still readable) and the whole stuff requires GEM as graphical system (yes, I am still keeping my first computer, an IBM XT clone, somewhere in my parents' place). So much about the knowledge if have obtained so far. The machine powers up, the spectral lamps don't start without software control as it seems, and I'll need to glue a mirror back into its bearing (Luckily, it didn't break into pieces when it fell off).</p> <p>I would like to ask if there is anyone familiar with this crazy item, and might sacrifice her/himself for answering some questions as they might occur during the resuscitation process.</p> <p>Actually, my goal is to run the whole stuff in a virtual machine on a Linux system, as all the control software needs to access, is a serial port to control the instrument and to read the data.</p> <p>Many thanks for your help!</p> <p>Wolfgang </p>

http://www.majorgroove.org/questions/274/prediction-tool-for-antigenic-peptides WS 2010-06-10T13:10:34Z 2010-06-10T13:10:34Z

<p>Dear Experts,</p> <p>I plan to order some custom peptide antibodies. While I have found a useful resource to estimate the antigenicity of peptide sequences, Antheprot (http://antheprot-pbil.ibcp.fr/), I wonder if there is a tool that I can use to check whether a selected sequence is present somewhere else in the species' proteome. Of course, one may BLAST each possible peptide manually, but maybe there's an automated solution? I have come across <a href="http://peptideselect.invitrogen.com/peptide/index.jsp" rel="nofollow">http://peptideselect.invitrogen.com/peptide/index.jsp</a>, unfortunately, it seems not to be functional (Opensuse 11.1 / Firefox 3.5.9 or W2KSP4 in a VirtualBox).</p> <p>Another question: How many AA is most appropriate? </p> <p>Thanks for your help!</p> <p>Wo</p>

http://www.majorgroove.org/questions/242/pcr-primer-dimer-formation unknown (google) 2010-04-09T09:28:53Z 2010-05-29T00:06:16Z

<p>Hi all. I seem to have a serious problem with primer dimer formation. I am not sure but I believe it is for this reason that my PCRs aren't working lately. Is there anything I can do to completely get rid of them while not compromizing the product? I need my products so bad but the bands are either faint or absent from my agarose gels. please help</p>

http://www.majorgroove.org/questions/260/help-with-lipid-micelles Rappley 2010-05-05T19:47:47Z 2010-05-28T20:43:37Z

<p>Hi all,</p> <p>I need to make lipid micelles for an experiment. My lipids are dissolved in chloroform. I dry them under argon, resuspend in aqueous buffer (10mM sodium phosphate, 100mM KCl, 1mM EDTA), vortex 1 min to get a cloudy solution, and then sonicate. I use a bath sonicator made by Laboratory Supplies Company Inc, as recommended by Avanti Polar Lipids. My protocol suggests sonicating for about 10-20 min.</p> <p>I know that the solutions are supposed to get clear (or at least, less cloudy) following sonication and that this change indicates that micelles have formed. However, my solutions remain cloudy even after 45 min of sonication. My questions are:</p> <p>1) What can I do to get micelles to form?</p> <p>2) How can I verify the presence of micelles? Could I use a quick measurement (light scattering? CD? turbidity?) to verify that the micelles have formed properly?</p> <p>Thanks in advance!</p>

http://www.majorgroove.org/questions/269/find-the-best-model-for-molecular-replacement Paul Lindblom 2010-05-23T17:35:37Z 2010-05-27T23:33:51Z

<p>Hi everybody,</p> <p>I just crystallized a new project protein. How can I find a possible model for using molecular replacement? I have the sequence of my protein. Is it enough to make a sequence search in the pdb? Or is there another approach I can use?</p> <p>Thanks a lot,</p> <p>Paul</p>

http://www.majorgroove.org/questions/239/ligand-fitting-with-coot Neha 2010-04-08T06:10:21Z 2010-05-23T23:23:42Z

<p>I have been trying to fit the ligand in electron density since a long time. I could place it in the right place by simply using 'rotate/translate zone'. Now i am facing difficulty in rotating a single bond of the ligand to achieve different conformation. When i opened mmCIF file of that particular ligand, coot gives a message 'CIF dictionary contains no restraints!'. What should be the solution to this problem?</p>

http://www.majorgroove.org/questions/264/camkii-or-other-pyramidal-cell-antibody-for-fixed-frozen-mouse-brain-sections unknown (google) 2010-05-21T01:10:52Z 2010-05-23T04:44:45Z

<p>What is a good antibody and staining protocol for camKII? Is there another marker for pyramidal cells that has a good antibody? I am staining mouse brain, fixed frozen tissue. I would like to ID pyramidal cells in the basolateral amygdala.</p>

http://www.majorgroove.org/questions/265/primer-design-for-splice-variants Rosh 2010-05-21T13:26:17Z 2010-05-22T10:49:27Z

<p>I have been trying to design primers for the spice variants. How can I design exon specific primers to identify the spice variants? There is no information on the number of exons in the NCBI website. Ensembl shows two variants and they have different exons. How can I design primers in this situation? Would be great if anyone can help me in this matter.</p>