Very interesting--I’d like to hear more about this heat-shock idea. First, am I understanding correctly? Grow at 37 deg to desired OD; heat shock 20 min; then chill and induce/express at desired temperature (e.g., 20 deg)?
Second, what sorts of problems did this help with? E.g., a lack of high-level expression, or high-level expression, but in inclusion bodies? Also, I second David’s post about expressing at reduced temperatures; this is de rigueur for us. Cheers, Pat --------------------------------------------------------------------------------------- Patrick J. Loll, Ph. D. (he, him, his)* Professor of Biochemistry & Molecular Biology Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102 USA (215) 762-7706 pjl...@gmail.com pj...@drexel.edu <mailto:pj...@drexel.edu> __________________ * screw you Donald T > On Mar 11, 2025, at 9:35 AM, Jon Cooper > <0000488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > > Hello, we found that a short (20 min?) heat shock to 42 degrees C followed by > cooling on ice prior to induction and growth at lower temp, as suggested by > David, worked for several proteins, but not all. > Best wishes, Jon Cooper. > Emeritus at UCL. > jon.b.coo...@protonmail.com > Sent from Proton Mail Android > > > -------- Original Message -------- > On 11/03/2025 11:18, Rafael Marques wrote: > Hello folks, > > I have been trying to purify a GST-tagged protein for a while but the results > are far below the expected. The majority of my sample is in the pellet > fraction and the yield of my expression is very good. This is known because > the protein was identified both by Mass Spectrometry and Western-blot. I > believe my protein is laying inside inclusion bodies. I have tried to > diminish the IPTG concentration, express it for a shorter time and also > different E.coli strains but the results are still the same. As a last > resort, I was thinking about refolding. I found this paper (attached), from > 2004, but I was wondering if someone who has experience regarding protein > refolding could shed some light on this topic, principally if there are more > updated protocols. > > Best wishes > > P.S. His-tag did not improve my results too > > > ______________________________________________________ > > Rafael Marques da Silva > PhD Student – Structural Biology > University of Leicester > > Mestre em Física Biomolecular > Universidade de São Paulo > > Bacharel em Ciências Biológicas > Universidade Federal de São Carlos > > phone: +44 07861 273773 > > "A sorte acompanha uma mente bem treinada" > ________________________________________________ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 Patrick Loll pjl...@gmail.com ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
