Protein standard curves - (May/18/2010 )
Hello, I am going to quantitate my protein with either the bradford method or the BCA method and you have to make a standard curve of known protein concentrations. Apparently you are supposed to do this standard curve every time you quantitate protein. Why can't you just take measurements for a standard curve with BSA once in your career and use it every time you want to determine an unknown? If I make a BSA standard curve one day, why would it be any different on another day?
Both the BCA and bradford are colorimetric detection systems that rely on a quantitative reaction with the protein. With increasing time and temperature, the reagents oxidise (BCA) or the reaction changes pH (Bradford) or the reaction goes to completion, which is not what you want. Minor variations in the incubation time of the assays results in colour changes that are detectable by spectrophotometer.
Basically I am saying that if you vary the time (or temperature) of incubation you will have different readings out of the spec, which will not be comparable with ones done at a different time or temperature.
Personally I would go for the BCA, much more stable reagents and wider reference range.