I'm trying to purify a protein from the cell lysate using single-chain antibodies immobilised on NHS-terminated magnetic beads. The yields are pretty good, however I get a lot of background proteins (mostly low MW) -- no matter how many washing steps I do. After antibody immobilisation the beads are being blocked with 3 M ethanolamine (as the protocol suggests), so no extra proteins should stick to these. But even with the empty blocked beads incubation gives the background. What could be the reason for that and how to overcome the issue? I see the publications of other people using that protocol and it works like a charm. Many thanks for your time and answers!
There must be some non-specific interaction most likely between proteins and the magnetic particles. What brand are you using?
@Omar I'm using these ones http://www.bioclone.us/N-hydroxyl-succinimide-activated-magnetic-beads-particle-resin-matrix.html from Bioclone Inc.
@Dominique no, this protocol is not requiring BSA blocking strictly. The non-specific sites should be blocked with 3M ethanolamine (presumably). We used amine-terminated magnetic beads before -- went bad, since we had more BSA in our eluate, than the tagret protein. Which is not very acceptable. And background proteins appear with those as well together with BSA and target.
Well, ethanolamine (just like BSA if used at the same blocking step) is just reacting with residual intact NHS but doesn't prevent from non specific binding of proteins at the bead surface. I think it is necessary to saturate the beads with BSA at the storage level as mentioned in the leaflet (excess should be washed off by several washes with PBS prior affinity purification).
@Dominique wouldn't the non-specific binding to BSA happen then? To my knowledge it's pretty sticky to everything...
That's the purpose : after BSA saturation and washes, only specific interaction (in your case Ag/Ab) should displace the BSA leaving the other non specific sites on the bead occupied by BSA (preventing non specific binding from your sample). As long as the elution step doesn't release BSA should be OK.
Yeah, but apparently proteins can more or less non-specifically stick to BSA itself -- like a sandwich. And apparently it does go off significantly in the elution. Did you have the same issue?
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