Prevention of Non-Specific Protein Binding to Beads

[kiki06]

Dear all,
how are you doing?

I have a question. I am trying to pull down a specific protein using beads.

My problem is I am getting a lot of non-specific proteins binding to the beads like plasma proteins.

How can I prevent this?

Thank you

[danfive]

Invest in a major protein depletion kit--albumin immunoglobulin etc. Also if endogenous antibodies are binding to protein a/g then treat the prep with pure protein a/g beads--centrifuge beads out of solution take sup and add to regular column with capture antibody coupled beads. Dump the first set of beads.

Cheapest way--but not 100% efficient--is to wash your beads 20-30 times with wash buffer, instead of the typical recommended 4-8 times.

[danfive]

Originally Posted by danfive

Cheapest way--but not 100% efficient--is to wash your beads 20-30 times with wash buffer, instead of the typical recommended 4-8 times.

This actually does work but if using plasma/ serum, you will most likely elute albumin, even after 30 washes. My guess is that it does dilute between the 1st wash and the 30th, but is never completely washed out. If your protein is in a different size range--you will be fine to co-elute albumin.

In biomarker work we are actually capturing albumin in plasma and looking for rare proteins that are stuck to it as potential biomarkers.