After lysis of cells, it is important to determine the total protein concentration of the sample. Accurate quantitation of the sample will allow you to load the proper amount of protein in each lane. This avoids overloading the lane but still allows adequate detection of the protein of interest. Proper quantitation is also critical when performing semi-quantitative or quantitative Westerns. For accurate determine of relative amounts of protein expression, the same amount of protein must be loaded into each lane.
There are several different methods for quantifying protein concentration in samples. Some (e.g. measurement of nitrogen content or radioactively labeling cells) do not rely upon absorptive properties of the protein sample. While these can be sensitive and accurate assays, the more commonly used assays rely on spectrophotometric determination of protein concentration and these will be the focus of this guide.
The different capabilities and limitations of several assays are discussed below.