Bio-Rad Protein Assay Dye Reagent Concentrate

The Bradford method is a common means for measuring the protein concentration in almost every molecular biology lab. I have been using the Quick Start™ Bradford reagent for the past 8 years to determine the concentration of proteins with different natures, molecular weights and concentrations. It’s quite versatile, with some limitations regarding the buffer conditions. Although there are many variations of the recommended protocol depending on sample volume and the concentration range of the protein to be measured, I have almost always used it in a 1 ml format (microassay procedure) with an 800 ul sample volume plus 200 ul concentrated dye reagent. The microassay procedure works in the linear range of 1.2 to 10 ug/ml (BSA) or 1.2 to 25 ug/ml (IgG). There is a standard procedure which involves 100 ul sample volume and 5 ml of diluted dye reagent and works in the linear range of 0.2 to 0.9 mg/ml (BSA) or 0.2 to 1.5mg/ml (IgG). There is also a microplate assay, a variation for lower sample volumes.

Addition of the protein sample to the reagent results in the formation of a blue colored product. The product formation can be analyzed and quantified by making spectrophotometric measurements at a wavelength of 595 nm. There are NO incubation steps and the resulting product is stable for several hours. This makes life much easier, especially if large numbers of samples are to be processed. There are convenient formats which allow the use of test tubes or microfuge tubes or microplates depending on the sample volume and concentration. I have worked by pipetting directly into the measuring cuvettes. There is a need to generate the standard curve with bovine serum albumin (BSA) or immunoglobulin G (IgG) depending upon the nature of the protein. Disposable plastic cuvettes, also available from Bio-Rad, come in handy when using the microassay procedure.

The problem with this reagent is its incompatibility with several other chemical components that are generally present in protein solutions (e.g. imidazole). There is a list available in the instruction manual that tells about these restrictions. I found it particularly difficult when I had to measure the concentration of proteins eluted in imidazole concentrations in excess of 50 mM. I had to either dialyze the samples against some other buffer or use an alternate protein assay kit. Another option was diluting the sample to a concentration that could be assayed. I have tried all 3 options depending on the availability of the protein sample. Diluting the sample was easiest because even dialyzing causes some dilution. When either dialysis or dilution was not an option, I used Pierce BCA kit for quantification. Another minor problem is the viscosity of the reagent as it makes accurate pipetting a problem.