HELP estimation of protein in ammonium sulphate purified igG - (Oct/08/2007 )
i want to estimate the amount of igG OBTAINED AFTER AMMONIUM SULPHATE PURIFICATION OF GOAT -ANTI MOUSE IGg. please let me know some methods to estimate the igG CONCENTRATION.
SDS-PAGE stained for proteins with quantitative dye and calculation of IgG bands (light and heavy chain) from densitometry, expressed as percent of IgG in (known amount of) protein or comparing with (known amount of) IgG standard (if you have one that is).
i have tried estimating by lowry assay. i have estimated the concentration by nanodrop. ammonium sulphate interferes with lowry. can we estimate by lowry? i have dialysed the sample and estimated the concentration. the concentration of igG ESTIMATED BY LOWRY WILL IT BE RIGHT?
I thought you want to estimate amount of pure IgG in your preparation, not just amount of protein... Dialyze your antibodies for at least 24 hours at 4 degrees against PBS then make any protein assay you like e.g. Lowry, BCA etc.
you can tca precipitate your protein (just enough for protein determination) and perform the lowry the way it was originally done.
you can also use the bradford method if your ammonium sulfate is under 1M.
I think K.B is onto your answer. Run a simple SDS-PAGE with say 1,3,5 ul of your purified product up against a serial dilution of a commercial antibody that tells you the concentration. Coomassie stain the gel and then compare the intensity of the heavy or light chain of your prep to the known. I'm not absolutely sure and asking around the lab has given different answers but you may want to make sure that the known antibody is of the same IgG type as your purified. This won't give you an exact measurement but will allow you to do a good estimation.
It is well known that A(280) (IgG( 1mg\ml) =1.4. So all you need are two cuvettes (1cm) ( one for control sample without IgG) and fotometer to do this measurement.