We use the Pierce BCA protein assay with a Bovine Gamma Globulin standard. We have found that this assay, when properly performed, gives the most reliable results of any we have evaluated.
Some antibodies recognize an epitope that spans secondary or tertiary protein structure.
The epitope is recognized by the antibody as long as the protein is not unfolded to a linear or semi-linear form. In western blot, the proteins are linearized by the SDS that is present in the separation buffer and do not refold correctly during transfer to the nitrocellulose or PVDF membrane. This causes a secondary or tertiary epitope to be distally separated and the antibody will not recognize the individual pieces. An antibody epitope may be formed by the close association of the GA of one end and the LT of the other. Removing the SDS during transfer with an excess of methanol does not guarantee proper refolding.