Protein Assay Kit From Bio-Rad

The vast majority of researchers in life sciences deal with protein solutions every day. Accurately determining the concentration of a protein in a solution can be critical for the outcome of an experiment and absolutely essential for its reproducibility from day to day. Failure to precisely calculate the concentration of a protein in a solution can lead to great delays, poor reproducibility of results and finally to poor interpretations of the available data. One of the fastest and easiest ways to determine the concentration of a protein solution is to use reagents that change color according to the concentration. A very popular reagent used for such purposes is the Bradford reagent.

Bio-Rad offers a protein assay reagent that can be used to determine protein concentration. This assay is based on the Coomassie Brilliant Blue G-250 dye that changes color in response to various concentrations of protein. The dye binds mainly to basic and aromatic amino acid residues along the polypeptide chain.

I have used this reagent diluting it 1:5 in distilled water (the recommended dilution is 1 part Dye Reagent Concentrate with 4 parts distilled, deionized (DDI) water) and when the assay was performed using the appropriate control samples, the results were reproducible. The whole process is very simple and when performed correctly, reliable results are guaranteed at the end of the day. Briefly, the protocol I follow begins by preparing the dilution (1:5) with distilled and deionized water. Then I mix 1 ml of the Protein Assay Reagent with 20 ul of 5 BSA solutions with predefined protein concentrations. Hence, I end up with 5 solutions of Protein Assay Reagent that contain 2, 5, 10, 15 or 20 ug BSA. In order to be successful, you must be extremely careful with your pipetting and of course, the concentration of the standard BSA solution must have been strictly defined. In order to increase the accuracy of these measurements, each one of the 5 BSA-Protein Assay Reagent solutions can be prepared in triplicate. These solutions are used to generate a standard curve that will demonstrate the correlation of the optical density of the protein assay solution at 595nm, with the concentration of the protein it contains. I commonly get curves with R²=0.99 (linear regression must be performed here with a software such as excel, minitab or SPSS) which is a strong indication of the accuracy of the curve. Finally, I add an appropriate volume of my protein solutions (it is best not to exceed 20 ul) in 1 ml of the diluted Protein Assay Reagent and I record the OD at 595 nm. I have mostly used 96-well plates for recording the optical densities of my samples. The curve that results from the BSA measurements is described by an equation of the form Y=A*X. Y represents the absorbance of your sample at 595 nm. So dividing Y/A=X you get X which is the amount of protein in the tube with the protein assay. By extrapolating these results to your original protein solution you can easily calculate its protein concentration.

A drawback of this reagent is the fact that it is very toxic, so you must wear protective clothing when using it. Another disadvantage is that it can easily give false values if it is not prepared according to the recommended guidelines. Furthermore, you must always bear in mind that there is an upper limit of protein concentration above which, the assay fails to give reliable results. Also, in contrast with other procedures (such as measuring OD at 280 nm), the sample that is used for the measurement with the assay is destroyed and cannot be used further. However, Bio-Rad’s Protein Assay Kit is a very simple and fast way to determine the concentration of a protein solution. Finally, another problem that arises with this reagent is that is associates strongly with plastic, causing difficulties when trying to clean them after the assay is complete (using bulk ethanol helps really a lot).

Overall, I am very satisfied by this reagent and I strongly recommend it to any researcher who works routinely with protein solutions. The main drawbacks, which are its toxicity and the requirement for very accurate handling, are more than balanced by the speed with which it can determine the protein concentration of a solution once the technique is mastered.