Dear researchers, I was going to purify a antibody from rabbit serum first by IgA/G (THERMO#89980) purification, but after Elution steps, there were not antibody found in SDS-PAGE gel (See attach). I will be very glad if anyone may help me to solve the problem. Thanks a lot!
Hi there,
The antibody you want to purify might not bind to the immobilized proteinA/G (not the right isotype) as there is a lot of material in FT and Wash1...
Hi Sa,
I think Dominique is right-your Ab is not binding to the column. I don't know the MWs of the bands that you see on your PAGE gel but if it is denaturing and the sample is reduced you could assume the huge band is serum albumin then the two bands below this in the FT and wash could be your heavy and light chains.
For rabbit IgG a pure proteinA column might be better although the A/G should still work. Was the column well-equilibrated and the sample diluted correctly with the binding buffer?
You could keep for longer time binding and also load the beads onto the gel to check if there is binding, if there is binding then there could be a problem with your elution.
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