Micro BCA Protein Assay Kit From Thermo Scientific Pierce Protein Research Products

The Micro BCA Protein Assay Kit (Product No. 23235) from Thermo Scientific Pierce Protein Research Products is an adaptation of the BCA Protein Assay Kit (Product No. 23225) that is optimized for quantification of dilute protein samples. It has all of the benefits of the BCA protein assay. It is a detergent-compatible, bicinchoninic acid formulation for the colorimetric detection and quantification of total protein from dilute samples; The Micro BCA Protein Assay Kit is designed to quantify samples in the 0.5-20 µg/ml range while the standard BCA Protein Assay Kit is designed for a higher range, 20 -2000 µg/ml, according to the manufacturer. Like the standard BCA assay, the micro version utilizes bicinchoninic acid (BCA) to detect Cu⁺¹ formed by the reduction of Cu⁺² by protein in an alkaline environment. Chelation of two molecules of BCA with one cuprous ion (Cu⁺¹) forms a purple-colored reaction product that is visual. The strong absorbance produced by this water-soluble complex can be measured spectrophotemtrically at 562 nm and is linear with increasing protein concentrations. The Micro BCA Protein Assay Kit from Thermo Scientific Pierce Protein Research Products has one of the lowest sensitivity ranges of any comparable protein assay kits on the market. I have used this product to measure protein samples that were too dilute ( approximately 5 ug/ml) to be measured using most other commercially available protein assays that I am aware of.

The kit includes all of the reagents needed to perform the assay as well as the BSA used for the standard curve. These reagents will work with either of two procedures; a test tube (linear working range of 0.5-20 µg/ml) or a microplate (linear working range of 2-40 µg/ml). Both procedures have separately written procedures included in the instructions that come with the kit. The main difference between the procedures is that the test tube procedure requires a larger volume of sample (1.0 ml/test tube), incubation at 60°C for 1 hour, and measurement in a spectrophotometer. Whereas the microplate procedure needs only 150 µl/well, incubation at 37°C for 2 hours, and a microplate reader for measurement. There are sufficient reagents for 480 test tube assays, and 3,200 micoplate assays. Users must supply the test tubes or microplates and the cuvettes/spectrophotometer or microplate reader. My experience is with the microplate assay/microplate reader because I needed to measure very dilute samples.

The only disadvantage of the microplate procedure over the test tube procedure is that the linear working range is higher (2-40 µg/ml) than with the test tube procedure (0.5-20 µg/ml); this is because of a shorter light path length in microplate readers as compared to spectrophotometers. Furthermore, the linear range can be even higher. In some instances, when I used the microplate procedure, the standard curve was only accurate starting at about 5 µg/ml. Another potentially negative aspect of both the microplate and the test tube procedure for those who do not like calculating dilutions is mixing the reagents. Depending on the number of samples you have, it could be tricky to calculate a 25:24:1 ratio for the three reagents that make up the working reagent that is added to your samples. However, this may be trivial to some.

The best thing about the Micro BCA Kit is that it has one of the lowest linear working ranges of all the commercial protein assays that I’ve come across. This is a good choice when working with dilute samples. It also extends the use of the kit. Also, it’s highly beneficial when you have very small volumes of samples with little to spare. I take a concentrated sample and dilute it to within the working range of the assay. This works well with the microplate procedure because even though the linear working range is higher than with the test tube procedure, it requires a very small volume: 150 µl vs. 1.0 ml for the test tubes. Either procedure is much more sensitive than the original BCA Protein Assay Kit which has a sensitivity range of 20-2000 µg/ml.