I am expressing a protein 71 kDa in E.coli .After Ni-NTA purification it resulting 4-5 bands in SDS gel. After purifying through FPLC further also it is showing the same in SDS gel. I am unable to stabilize the protein by adding many stabilizers during cell lysis too. Need some suggestion
Hi there,
do the bands run lighter than full length protein?
do they coelute with full length in SEC?
If yes, these bands might result from mild proteolysis. Do you supplement the lysis buffer with protease inhibitor cocktail?
yes these additional bands are the fractions of full length proteins only..
I have tried adding protease inhibitor PMSF and Benzonase coktail for reducing the proteolysis but got the same output.
You shoud try a protease inhibitor cocktail which exhibits a broader spectrum of inhibition towards proteases. For instance Complete from Roche Diagnostics (see the following :http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/04693116001bul.pdf). For info benzonase is a nuclease in charge to destroy DNA during lysis and PMSF is only active on serine proteases.
i agree with the answers so far, Roche inhibitor tablets are the best but most expensive. AEBSF is at least better than PMSF, which has reduced stability in aequos solutions.
Are you using french press or a ultrasonic device for the lysis ? for the latter: reduce the time or amplitude.
All the best
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