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 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/
