Expression in E.coli
Posted 12 September 2010 - 08:14 AM
harvest 100 ml culture
re-suspend pellet in 5ml lysis buffer, sonicate
supernatant: soluble fraction
pellet: re-suspend in 500 ul lysis buffer: insoluble fraction
Use 1-2 ul of the insoluble fraction for loading on PAGE
I add regular PAGE loading buffer (which contains DTT, a protein denaturant) to all (soluble/insoluble) samples and heat them at 100C for 2 min.
The combinatorial effect of DTT and heating denatures and solubalise the protein/s in inclusion bodies
Posted 12 September 2010 - 10:56 PM
And dont u add chaotropic agents like urea, etc to the insoluble fraction?
500ul of which lysis buffer u use to add to the insoluble fraction,
whats ur lysis buffer composition?
Edited by bhavana, 12 September 2010 - 11:00 PM.