Protein estimation directly from SDS-PAGE loading buffer for standardization of samples from cell lysates or tissue homogenates before Western blot analysis.

Western blot (5) is a powerful tool to study the regulation of specific proteins from cell cultures or tissue extracts. The standardization of the total protein content is an essential step before the analysis of specific proteins regulated by different factors in cells or tissues. Standard protein assay procedures (1,4) based on the colorimetric reactions are often affected by the concentration of detergent, salt, thiol-reducing compounds, chelating agents, lipids, or other components of homogenization buffers. The current methods used to estimate protein content before western analysis therefore limit our ability to compare protein expression in samples of different origin, cell culture, or tissue. Here, we describe a simple and reliable method for quantitative determination of total protein directly from the sample loading buffer commonly used for electrophoresis (2). The proposed method of quantification works in two steps, combining two procedures published previously; extraction of proteins by methanol-chloroform-water (6), followed by staining of protein spots on nitrocellulose membranes (2). The extraction allows the recovery of soluble and hydrophobic membrane proteins from homogenization or lysis buffers. Extracted proteins are then dissolved in the electrophoresis loading buffer and spotted on a nitrocellulose membrane to estimate protein concentration with amido black 10B before loading the sample on SDS-PAGE gel (2,3). In the present report, we have applied the procedure described above to study the expression of prostaglandin H synthase 2 (Cox-2) in cell culture by western blot analysis (Figure 2B). Endometrial cells cultured in 24-well plates were grown to confluency and washed twice in PBS, and 200 μL lysis buffer [10 mM Tris-HCl, pH 7.4, 1% SDS, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonylfluoride (PMSF); Sigma, Mississauga, ON, Canada] were added to each well. The cell lysate was transferred to a microcap tube, and protein extraction was performed as follows: in a microcentrifuge tube (1.5 mL) containing 200 μL lysed cells, 600 μL methanol, 200 μL chloroform, and 500 μL water were added, and the tube was vortex mixed vigorously and centrifuged for 3 min. The upper phase was removed carefully with a handheld pipet and discarded, keeping the interphase (containing the precipitated proteins) and the lower phase in the microtube. A further 800 μL methanol were added to the rest of the microtube for protein precipitation, and the vortex and centrifugation steps were repeated. The supernatant was discarded and the protein pellet dried using a SpeedVac (5 min). The proBenchmarks