Alternatively, why not do a quick polish step using a long gradient on a 5 mL anion or cation exchange column, followed by a gel exclusion cleanup if necessary? Once the proteases have been removed from the crude extract, further degradation of purified protein should be minimal. This purification treatment routinely works for us with proteins at this contaminant level.
Roger Rowlett On Feb 15, 2012 8:19 AM, "Sivasankar Putta" <[email protected]> wrote: > 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 > > > >
