how to efficiently elute protein from Protein A-agarose beads? - (Jul/22/2007 )
Hi everyone,
I am doing some metabolic labeling and IP using monoclonal antibody. I used Protein A-agarose from Roche for IP. My problem is the elution step. As I need to do an enzyme assay for the IPed protein, so I am using Tris+SDS (ph 7.4) to elute the protein from agarose beads by boiling at 100'C for 3 to 5 mins. But the efficiency is low. The beads are still hot, and the eluted solution is not hot, with only hundreds cpm.
Anybody can give me some suggestions?
Thanks very much!
I am doing some metabolic labeling and IP using monoclonal antibody. I used Protein A-agarose from Roche for IP. My problem is the elution step. As I need to do an enzyme assay for the IPed protein, so I am using Tris+SDS (ph 7.4) to elute the protein from agarose beads by boiling at 100'C for 3 to 5 mins. But the efficiency is low. The beads are still hot, and the eluted solution is not hot, with only hundreds cpm.
Anybody can give me some suggestions?
Thanks very much!
Sorry, I don't understand motivation for your elution method. Why not to elute with 0,1 M glycine and then separate Mab and antigen on GF in glycine containing buffer? Your procedure very harsh and also in eluate you will have Ab Ag mixture.
you mean elute in 100mM glycin pH3.0? Then add neutraliztion buffer (1.0 M Tris, pH 8.0)?
I am doing some metabolic labeling and IP using monoclonal antibody. I used Protein A-agarose from Roche for IP. My problem is the elution step. As I need to do an enzyme assay for the IPed protein, so I am using Tris+SDS (ph 7.4) to elute the protein from agarose beads by boiling at 100'C for 3 to 5 mins. But the efficiency is low. The beads are still hot, and the eluted solution is not hot, with only hundreds cpm.
Anybody can give me some suggestions?
Thanks very much!
Sorry, I don't understand motivation for your elution method. Why not to elute with 0,1 M glycine and then separate Mab and antigen on GF in glycine containing buffer? Your procedure very harsh and also in eluate you will have Ab Ag mixture.
Yep, I agree with circlepoint: elute with 100mM Glycine pH 3 then neutralize with Tris-HCl pH 8. That should do the trick...
wang proposes slight different elution : with 100 mM glycine at pH2.5. Eluents neutralized with 1.5 M Tris HCl at pH 8.8 or by dialysis against BC100 for 3 hr to renature proteins.
thanks to everyone.