I have a problem with the step of the crosslinking of the resin-GSH to GST-protein, in the contest of an antibody purification.
A portion of my protein is fused to GST in the N-terminus and I purify the recombinant protein from BL-21 E. coli strain. During the incubation of the lysate with the resin-GSH, the resin itself aggregates in insoluble particles of diverse size. Adding 10mM of DTT solves the problem, but during the crosslinking, I’m in trouble again and re-adding DTT (even 50mM) doesn’t help. The crosslinking steps are as follows:
1. Wash the protein-bound resin 2 times with Na2B4O4 at RT.
2. Resuspend the resin in Na2B4O4 with 20mM of DMP, 45 minutes rocking at RT.
3. Wash 2 times with etanolamine 0,2M pH 8 (!! The resin aggregates again!)
4. Resuspend in etanolamine 0,2 M pH8 for 2 hours rocking at RT.
The last step should be a wash with PBS.
Has anybody of you ever had this kind of problem? Any suggestion, anyway?
Thank you in advance.