Solubilization and Assay of Cellular and Tissue Protein

Abstract

Solubilization and accurate quantification of cellular protein are central to analytical methods such as Western analysis. Alternate homogenization buffers can be used according to need, but must match the assay used. The Bio-Rad (Hercules, CA) assay, based on the method of Bradford (1), is faster, but is not able to tolerate SDS although it can tolerate β-mercaptoethanol. In contrast the bicinchoninic acid (BCA) assay (2) has greater linear range, and has greater tolerance for SDS but not β-mercaptoethanol. However, there is also less tolerance for a large sample volume. Salt buffers can also be used with PMSF, leupeptin, or aprotinin added in each assay, as long as the interfering reagents are omitted as appropriate. In this chapter, procedures are described for both assays, scaled for use with a microtiter plate and plate reader. If a plate reader is unavailable, however, the same assays can be run in a visible-wavelength spectrophotomoter, but with all assay/sample volumes scaled accordingly to match the cuvet volume used.