Quantifying Protein in 2-D PAGE Solubilization Buffers

Accurate quantitative and qualitative comparison of resolved proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) requires that identical amounts of sample be loaded on each gel. Use of trichloroacetic acid (TCA) or organic solvent precipitation protocols followed by a protein assay results in either over- or underestimation of solubilized protein concentration. Furthermore, the practice of loading equivalent amounts of radioactively labeled protein-based counts/min or loading extract from the same number of cells is inaccurate. The author has observed that radioactively labeled cell extracts exhibit 10–20% variances in protein concentration. Similarly, solubilization of 2�10⁶ cells from a number of colorectal carcinoma cell lines resulted in protein concentrations ranging from 1.60 to 3.93 μg protein/μL buffer (1 ). Therefore, the ability to quantify protein actually solubilized in 2-D PAGE sample buffers is necessary to allow users to adjust for variable solubility properties of protein(s).