The BCA™ Protein Assay Kit is used for detection and quantification of total protein. Reduction of Cu
2+ to Cu
1+ by protein in an alkaline medium is detected via colorimetric detection using a reagent containing bicinchoninic acid (BCA). A purple colored water-soluble complex is formed by the chelation of two molecules of BCA with one cuprous ion. This complex has a strong absorbance at 562 nm. Each kit includes BCA™ Reagent A (500 ml), BCA™ Reagent B (25 ml) and Albumin Standard Ampules (2 mg/ml, 10 ampules). All these contents can be stored at room temperature.
I have used this kit for protein measurements using microplates, which require small volumes of protein samples (10-25 ul). According to the assay protocol, test tube procedures can also be done with larger volume (0.1 ml) of protein sample.
Protein concentrations are generally determined by comparison with a reference standard of protein, such as bovine serum albumin. A series of standard dilutions are prepared and assayed along with the unknown(s). The content of one Albumin Standard (BSA) ampule is sufficient to prepare a set of diluted standards. I follow the company-provided protocol for the dilution of the BSA standard. A working reagent is prepared by mixing 50 parts of BCA™ Reagent A with 1 part of BCA™ Reagent B. This working reagent is stable for a few days when stored in a closed container at room temperature. Either standards or unknown samples (25 ul) are added to the microplate-wells followed by the addition of 200 ul working reagent. The plate is covered and incubated at 37ºC for 30 minutes. The plate is then kept at room temperature and the absorbance is measured at 562 nm. The absorbance of the blank standard replicates is subtracted from the measurements of all standards and unknown sample replicates. A standard curve is prepared by plotting the average blank corrected measurement (at 562 nm) for each BSA standard vs. its concentration in ug/ml. The standard curve is used to determine the protein concentration of unknown samples.
Overall, I have been using this kit for the last 1 year I am very happy with this kit. The results are consistent in replicates while using microplate-procedure.