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
