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Purification of an antibody: crosslinking of resin-GSH to rGST

GST purification resin-GSH crosslinking

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#1 Moonila

Moonila

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Posted 20 May 2014 - 04:32 AM

Greetings,
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.







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