CNBr -Sepharose 4B

graeser at ubaclu.unibas.ch graeser at ubaclu.unibas.ch
Tue May 9 11:20:46 EST 1995


In article <3nogoq$p25 at mercury.hgmp.mrc.ac.uk>, <Unknown> (anonymous) writes:
> I am looking for some help in coupling a poorly soluble protein to CNBr- 
> activated Sepharose 4B.    We want to affinity purify some antibodies, but
> the antigen (a recombinant protein) is only soluble in guanidine HCl  or
> urea.   When diluted at least 20 fold with phosphate, the protein remains in 
> solution, but at these concentrations the coupling efficiency will be very low.
> 
> I was wondering if anyone has any experience of coupling a protein as a
> slurry, perhaps in the presence of a non-ionic detergent.   Alternatively,
> is there an affinity matrix which is compatable with solvents containing
> amino groups.
> 
> 
> Thanks in advance
> 
> Pete Jenner
> 
> Dept of  Mycobacterial Res.
> NIMR.
> London
> UK.

Hello Pete,

try to dialyze your protein against 0.1M NaHCO3 / 0.2M NaCl / 0.1% SDS. It will
precipitate in the dialysis bag and solubilize again, this takes about 36 hs.
You can proceed with the coupling reaction in this buffer as indicated in the
product sheet. The washes after coupling will be first two times with coupling
buffer containing SDS, then with the normal buffers as indicated in the product 
sheet. We are doing that with several insoluble proteins and have good success.

			Good luck
     			           Ralph
 








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