I typically use the BCA assay method to determine the concentration of protein samples. The protein that I am currently studying tends to aggregate and, after following an approach described in a paper, I found that incubating the samples in DTT immediately reduced the amount of oligomer (i.e. protein aggregate) on a Western blot. The only problem was that the BCA assay is not compatible with DTT and the samples immediately turned dark purple, making them impossible to assay via the BCA method. The answer was not as simple as switching to another assay reagent because some other methods are not compatible with the SDS in my cell lysis buffer. It was clear that I was going to have to precipitate the protein or dialyze it, but I did not have the appropriate reagents in the lab; in addition, I did not want to add hours to my protein assay procedure. That is when I discovered the Compat-Able reagents from Pierce. These reagents precipitate the protein in about 10 minutes, removing the substances that interfere with standard protein assays. The procedure is simple and if you are unable to perform the protein assay right away, you can store the pellets in the refrigerator.
One small disadvantage to the Compat-Able reagents is that the standards also have to be treated with the reagents in order to accurately measure the concentration of the protein samples. However, the procedure only involves mixing the samples with reagents and centrifuging them, making it easy to process several samples at once. Also, the protein is precipitated and must be resuspended in ultrapure water or working solution. As with any precipitation, if the pellet is not thoroughly resuspended by vigorous vortexing, the protein concentrations measured in the BCA assay could be incorrect. It is preferable to perform the BCA assay in the same tube so no sample is lost in its precipitated form. Resuspending the pellets in working solution instead of water may be helpful as well.
The reagents can be purchased together with Pierce’s BCA or Coomassie Plus Protein Assay Kits or separately; the price should be within the budget of most labs. Pierce has also introduced a BCA assay reagent that is compatible with reducing agents at the concentrations used in most protein sample buffers. This product may be a better choice for some applications where DTT is limited to 5 mM or beta mercaptoethanol to 35 mM. I added as much as 30 mM DTT and was able to precipitate it out with the Compat-Able preparation reagents and perform a BCA assay. The reagents provide an added bonus of removing other substances that could potentially interfere with the protein assay, such as detergents and salts.
Compat-Able Protein Assay Kit From Pierce
A simple way to remove reducing agents that interfere with BCA and other protein assays.
If the precipitated protein is not properly resuspended, the assay results could be inaccurate.
The Bottom Line
The procedure only takes a few minutes, so it is definitely worthwhile for anyone who wants to perform a BCA assay on samples that contain interfering substances.