I have been having problems in doing the bradford ptoein assay for quantitation of hyek 293 lysates. I dilute the lysates 30x (5 ul in 150 ul) and then use 20ul of diluted lysate with 180 ul of biorad protein assay (bradford reagent diluted 1:4 with water in a 96 well plate. I do a standard curve of between 0 - 175 ug/ml of BSA.
I get very inconsistant results, the duplicates are around 20% different and if I repeat the assay the inaccurancies are larget, e.g. if in assay 1: A = 1 mg/ml and B = 1.2 mg/ml in assay 2 it might be reversed and A might be 1.2 and B might be 1. I have triple checked all my steps and are very careful when doing them, but I cant seem to get rid of the inconsistancies, anyone can help?