Re: [ccp4bb] protein degradation during concentration for crystallization trials

Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to

[ccp4bb] protein degradation during concentration for crystallization trials

Dear all I am working on purification of 14 kd protein(pI  8.3, basic protein)  that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout

Re: [ccp4bb] protein degradation during concentration for crystallization trials

Hi, MBP tag: 1.there might be a TEV cleavage site in your MBP variant. 2. your protein needs salt to stay in solution 3. your protein forms aggregates with MBP GST tag: you probably concentrate a protease together with your protein. You need a protease inhibitor kit to take care of different