Re: [ccp4bb] protein stain, B-PER
Hi, if you use pLysS E coli strains and you're working with soluble cytoplasmic proteins, a couple of cycles of freeze-thawing in LN2 will break your cells pretty well with high protein recovery, without the need of extra exotic chemicals added to your mixture. Just put some DNAse to avoid excess viscosity of your sample and get rid of nonspecifically bound DNA. If the cells are not pLysS, the same method works fine if you add to your lysis buffer a bit of lysozyme and do a couple more cycles. Regarding protein stain, here's my suggestion for a 5-minute clean, efficient stain-destain protocol (with microwave, though): - after running your gel, put it in a glass beaker or even better in one of those low-profile glass containers used to make ice baths, filled with ~1 cm of deionized water - microwave the gel for 1 minute, max power (on my microwave, 1000W) -wash out the water, then add 40 ml of PageBlue. Cover your container with a paper towel (do not seal it), and microwave 3 times for 15 seconds at maximum power (1000W), gently shaking the container between the cycles. The blue stain should never boil. If you see that it starts boiling, reduce the length of the cycles. - collect back the blue stain solution, rinse the gel with water (again, 1 cm in the container), and microwave for 30 seconds. Change the water again 1-2 times. its done, low background, ready for imaging. In my hands, the whole procedure takes less than 5 minutes and gives beautiful gels. The PageBlue solution that we use (Fermentas) is much more sensitive than standard coomassie R250 and can be re-used an incredible number of times. This makes this staining solution cheap despite the cost of a 1L bottle (I am staining 3-4 gels per week and Ive been using the same 50ml tube of PageBlue for the last 3 months at least). Without the microwave, the same solution works well if you stain 10' and destain in water for about 1 hour. HTH, Federico Forneris, PhD --- Crystal and Structural Chemistry Bijvoet Center for Biomolecular Research - Utrecht University Padualaan 8 3584 CH UTRECHT - The Netherlands Tel. +31 (0) 30 253 2868 - Fax +31 (0) 30 253 3940 http://www.crystal.chem.uu.nl/group-gros/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas Edwards Sent: giovedì 15 marzo 2012 15:24 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein stain, B-PER Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse !). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, nondestain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne
Re: [ccp4bb] weird--No protein expression using pET30a
Hi Gerry, In one case, I had a mutation in the ribosome binding site sequence. If you haven't already checked, it might be worth checking whether the 5'- and 3'- junctions close adjacent to the ORF are all correct to make sure the transcriptional and translational elements do not somehow contain mutations. On occasion, figuring out what is wrong with the current construct might send one on a endless wild goose chase. So if a bunch of ideas with the current construct fail and you still want to try and express in the pET system before moving on to another system, I recommend the pET Expresso system. No need to ligate etc. Add clean PCR product + vector + cells and you directly screen for clones that are made by homologous recombination in bacteria. Cheers, Raji On Sat, Mar 17, 2012 at 2:26 AM, Jerry McCully for-crystallizai...@hotmail.com wrote: Dear ALL; Recently, one of my colleagues cloned a gene (200aa) into pET30a vectors with either a N-ter or C-ter His6 tag. The correct reading frame was confirmed by sequencing. However, it is weird that there was no protein expression either in the soluble fraction or as inclusion bodies. Could anyone give some instruction? Thanks a lot and have a nice weekend, Jerry -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] weird--No protein expression using pET30a
Hello, Probably a stupid comment on my part, but anyways, make sure your strain has a T7 polymerase! (I have forgotten before). And I agree with the last idea that sometimes it is worth to just start from scratch. New construct new vector. It may simply just work. HTH, Yuri
Re: [ccp4bb] GST-fusion protein production in insect cells
Sent from my HTC on the Now Network from Sprint! - Reply message - From: Sebastiano Pasqualato sebastiano.pasqual...@gmail.com Date: Fri, Mar 16, 2012 10:12 am Subject: [ccp4bb] GST-fusion protein production in insect cells To: CCP4BB@JISCMAIL.AC.UK
Re: [ccp4bb] GST-fusion protein production in insect cells
Hi :) In addition to excellent replies already posted, a few thoughts: 1. Do the whole-protein MS of your stuff. Assuming that you get a spectrum then a) If you are right and it is indeed your recombinant GST - you will see a mass pattern that's interpretable via analysis of probable cleavage sites. You will also glean some understanding of where the undesired cleavage (or premature termination!) occurs. b) if the suggestions of previous replies are right, then these protein(s) will be of the insect-cell origin. I have not seen this to be a huge issue in the past, however it may be due to the experience with Sf9, Sf21 cell lines and relatively low use of Hi5 in my past work. Switching a strain might help (maybe you already tried, and it still sucks, sorry). If you can't get a spectrum of the whole protein I would potentially suggest tryptic cleavage and peptide MS. You will get at least an idea whether (a) or (b) is more likely. In general (b) is tougher to fix because this means that your protein isn't being expressed in large enough amounts. You don't mention - is the insoluble fraction interesting? Is there a big fat band at the correct m.w. in the insoluble material? Perhaps a change of lysis conditions might help, or expression at lower temperature (yes, insect cells do grow at somewhat lower temperature, however they're not very happy there) or lower MOI. Also: do you *need* MultiBac system in your case? It's an excellent tool for large complexes but it's not clear from your post whether you're working with one protein or many? MultiBac has Cre/Lox leftovers in the bacmid which may be un-fun in your case for some reason (unlikely but...) Take a look at the DNA RNA structure (predicted) in the junction region. Is there any 'funny business' with hairpins, ribozymes, unusual structures of any kind? A cryptic ribozyme can really mess up one's life (it's not likely that you have one, much more likely an early termination structure or inefficient read-through). Splicing can also be difficult to control on occasion - any presence of strong splicing sites? AG/GURAGU and YAG/RNN with a few additional parameters thrown in. This would require a fair bit of RNA to be in between the sites, too. Cheers, Artem On Fri, Mar 16, 2012 at 7:57 AM, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Dear CCP4ers, Dr. Berger, we have an accumulating series of GST-fusion proteins here that are all displaying the same behavior when expressed in Hi5 insect cells with the MultiBac system. What we are experiencing is a massive production of free GST and only a limited amount of fusion protein. Since the linker we have engineered between GST and our protein is the one that exists in the pGEX-6p vector series, with the preScission protease site, I was wondering if any of you had experience of this cleavage site being processed within these insect cells. Thanks in advance for the help, ciao, Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990
