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