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protein estimation of monoclonal antibody - inconsistent values (Oct/17/2007 )

Hi all,
how do you do protein estimation of purified monoclonal antibodies
I do by bradford but the results vary a lot at different time points from the same aliquot
also, when I load approximately 2ug of antibody according to bradford on SDS-PAGE gel, i hardly see any bands on coomassie stain
can someone please tell me how do you do estimation of mabs
thanking you in advance


Dear Leelaram!

You needn't to do Bradford assay , only measure absorbtion of purified Mabs at 280nm and keep in mind that 1mg\ml have Abs(280) = 1.4

Very strange about absence of bands with 2ug of protein on SDS PAAG because limit of Coomasie staining is near 0.05ug\ul protein sample. So I think the main problem is your protein determination with Bradford? btw what protein did you use for calibration - BSA or gamma globulin?


and in what buffer is your antibody? did you blank for it?