Mab purification - no mouse IgG band seen (Oct/17/2007 )
I have recently purified a monoclonal antibody from 50ml tissue culture supernatant by protein G column. I have checked it's inhibitory activity against the respective antigen. But, to my surprise, when i checked the purified antibody on SDS-PAGE gel, i couldn't see any band corresponding to mouse IgG. but i could see the bands respective to bovine IgG which I guessed it by loading purified normal mouse IgG and also purified IgG from fetal bovine serum. Can someone tell me why am i seeing an inhibitory effect in the assay but no band of IgG even by silver staining.
all the suggestions are highly appreciated
thanking you in advance
may be there are more IgG from fetal bovine serum than your desired mouse IgG.
therefore the bovine IgG bound to the beads in the protein G column.
You may wish to use protein A column because bovine IgG binds poorly to protein A.
Hope this may help.
You could also culture your cells in serum free medium one or two weeks before collecting the supernatant for purification
thank you for your suggestions
even i was thinking of purifying by protein A
thank you once again