You can also try putting a different affinity tag on the other terminus, and use that as a second step.
JPK On Wed, Feb 15, 2012 at 11:25 AM, Xiaodi Yu <uppsala....@hotmail.com> wrote: > 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 > > > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *******************************************
