I'm trying to IP 82.6 kDa protein from excretory secreted antigen (ESA) fraction of the parasite in non-denaturing condition (for immunological assay). I am using 1mg of ESA fraction as input and incubate with monoclonal antibody followed by incubation with dynabeads. I do see a band in western blot when I use SDS as elution buffer.

But fail to see any band on using 0.2M glycine at pH 2.1. I am using 300ul of glycine buffer for elution (30 mins at RT with gentle rotation). I transfer the supernatant and add 200 µl of Tris-HCl 1 M, pH 7.5 for neutralization. I concentrate sample to 80-100ul which I used in Sypro ruby stained SDS PAGE gel/ western blot.

I have also tried other mild elution buffer like 0.2M citric acid, pH 2.1 and 3.5M MgCl2 as elution buffer but they also did not help.

Pls suggest how can I elute protein of interest. Thank you.