how to separate Antibody and Antigen in complexes
Posted 30 October 2012 - 05:21 AM
I have a sample from immunoprecipitation column - polyclonal porcine antibodies in complexes with salmonella proteins. (all in PBS)
any suggestion how do I separate them and isolate only salmonella proteins?
Of course there exist many IP kits with protein A or G on beads or stuff, but I'm looking for some simple solution to do this without any kits.
thanks a lot for your replies
Posted 23 July 2017 - 12:40 AM
In dissociation IC Elisa assay, we employed, for several biological fluids, dilute sample 1:100 (1 µl + 100 µl) with dissociation buffer (PBS buffer with 1.5% BSA and 0.2 M glycine-acetate), pH 2.5 and/or pH 3.5 to a 500 µl final volume and incubated for 20 min. at room temperature. The treated diluted sample is then pipetted into the sample reservoir of Microcon centrifugal filter device, YM-10 (10,000 mw cut-off by Millipore) and centrifuged at 8,000 × g for 20 min. at RT. The sample reservoir is then separated from the flow through, placed inverted into a second tube and centrifuged at 1000 × g for 3 min. at RT. The collected solution, containing the antibody dissociated from the antigen, is adjusted to pH 7.0 with 1 M Tris buffer, pH 9.0. The retentate volume is reconstituted to the initial volume (500 μl) with ELISA dilution buffer (PBS with 1.5% BSA and 0.1% Tween-20) and then dispensed in ELISA MTP at several dilutions to determine the antibody titer.