Hi Sivasankar:
Are you sure it is due to the protein degradation? Maybe you can try to do a
western blot or others to check if it is the product of degradation. By the
way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal,
maybe it is the truncation version of your protein.
After looking at the gel, it seems your sample was over-load or had lots of
unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final
concentration) and small amount of imidazle in the sample before you load onto
the column (for example, 20 mM Imidazole final concentration).
One small "trick" you can try is wash the cell with the buffer containing PMSF
once before lysising the cell.
Yu Xiaodi
Date: Wed, 15 Feb 2012 18:39:19 +0530
From: sivasankarpu...@iisertvm.ac.in
Subject: [ccp4bb] protein degradation
To: CCP4BB@JISCMAIL.AC.UK
Dear All,
Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi
domain) DNA binding protein, that we are expressing at 18 degree Centigrade in
E. Coli. The protein appears to degrade during purification; we have protease
inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and
1mM DTT throughout during purification ( right from lysis stage). We handle
the protein at 4 degree Centigrade.
Can you please suggest what precautions we can try to avoid such degradation ?
Please find the attached gel picture regarding protein
Sivasankar Putta
