Western Blot Protein Quantification Assays
After protein sample preparation, the next step of western blotting is total protein analysis. Total protein analysis is the process of quantifying the protein concentration. Protein concentration data is critical to getting good results on your blot: equal amounts of protein must be loaded into each well of the gel to avoid saturation issues, ghost bands, and other trouble. The concentration data produced by total protein analysis can also perform as a reliable loading control for semi-quantitative western blotting. Boster provides a range of high-quality protein analysis kits so you can get more accurate protein concentration data and better blots.
To quantify the protein using the BCA protein assay kit, the first step is to reconstitute the bovine serum albumin standard samples with 0.9% NaCl or PBS solution to create a solution with a known concentration. After preparing the BSA standard solution, create a gradient dilution using the BSA standard solution and the diluent solution included in the kit. Prepare the BCA working reagent using a 50:1 reagent A:B ratio and add it to the BSA gradient dilution. Incubate at room temperature and measure the absorbance of the samples at 562 nm. Plot the standard curve to determine the protein concentration of your protein sample.
For a detailed protocol for total protein analysis, see our Western blot handbook.
- Select the right protein assay for your application; choose between silver staining reagents, BCA assays, and commassie blue stains.
- Avoid inconsistent band strengths across wells, streaked bands, and saturation problems by getting accurate protein concentrations and optimizing loading amounts. See our troubleshooting guides for more information.