Hi, I'm interested in doing immunoaffinity purification of an insulin-like protein by using Elisa plates with bound antibody to insulin. I get a nice positive reaction using the sandwich method with a commercial plate and would like to bind my protein, wash off unbound proteins and then collect the bound protein . Can someone suggest an equivalent to an elution buffer that would separate the bound protein from the antibody attached to the well so that the protein can be collected pure?
Hi Arthur,
yes you can do this. You can try e.g. 0.1 M glycine pH 2.2 or 3 M MgCl2. Remember that the max. binding capacity for ELISA plates is only 400 ng/cm2 (and probably less). You may want to consider collect the protein from several wells. To keep the volume low, add the elution buffer to well 1, incubate for 30 seconds, then transfer to well 2 etc. In case you have enough sample you can use this approach with an 8 channel multipipette and collect the entire plate. Neutralize with NaOH if you use acidic elution buffer. If needed concentrate using ultrafiltration spin columns. You should apply the sample at a high enough concentration to saturate the binding sites of the coated insulin antibodies. If you can retrieve 50 ng / well you can get several microgram from a plate. So if it works well you can get enough for westernblot, protein gel and thus mass-spec analysis.
You can test if your elution buffer works efficiently simply by measuring the residual bound material after elution (simply wash after collecting the elution buffer and continue s usual. Compare eluted vs.non-eluted)
Good luck.
Thanks Markus!
Will the antibody come off also or is its attachment to the plate too strong?
Depends a bit on the buffer for elution. Binding to the plate is via hydrophobic interaction, elution at low ph shouldnt get the antibody off. Give it a try, if you run in problems you can precondition the plate.
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