Micro BCA Protein Assay Kit From Thermo Scientific Pierce

Pierce’s Micro BCA Protein Assay Kit is a wonderful addition to their protein modification, measurement (estimation) and manipulation group. It has taken the assay’s previous significant volume and quantity requirements and lowered the necessary amounts (concentration and protein) for determination. In the microtiter plate format, you can assay 25 -50 ul of your sample without any problem and get reproducible and accurate results.

The chemical reaction of “the BCA Protein Assay combines the well-known reduction of Cu²⁺ to Cu¹⁺ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu¹⁺) by bicinchonicic acid,” as stated in the protocols pamphlet. The assay is relatively simple to perform. First, prepare the reagent mixture: 25 parts of Reagent A, 24 parts of Reagent B, and 1 part of Reagent C. Then mix this mixture with your sample in a 1:1 mixture and pipette that mixture into either tubes or the wells of a microtiter plate and incubate for one hour at 60ºC. The tubes or microtiter plate are then read at 562 nm. The standards are evaluated for linearity, correlation and intercept. The unknowns are quantified from the linear curve.

The kit does a very respectable job with proteins that do not have any post-translational modifications (e.g. reducing sugars) which are known to interfere with most protein assays. This can be particularly detrimental when comparing protein estimations from more than one type of expression system or to the native protein source. I have found variations in results due to the amount of reducible sugar present. These are lot-to-lot variations; for example, BSA lots or protein from different preparations.

Pierce’s Micro BCA Assay is a consistent and reliable method for protein estimation. The assay is well designed and can tolerate a significant concentration of a number of potential interfering substances. However, the Micro BCA and BCA both have problems with reducing sugars. Proteins which have a higher percentage of glycosolation will have some difficulties with reproducibility, precision, and accuracy. Additionally, because there is some variability ratio of glycosylation to protein from lot to lot of protein purification, there will be observed differences between estimated protein concentrations.