Estimation of protein using Bicinchoninic acid (BCA)

This method was developed by Smith et al in 1985 and thereafter, Pierce Chemical Company exploited it by developing a kit. In this method, there is reduction of cupric ions to cuprous ions by the protein under alkaline condition. The cuprous ions are detected by using bicinchoninic acid (BCA). The macromoecular structure of the protein, the number of peptide bonds and the presence of four amino acids (cysteine, cystine, tyrosine, tryptophan) are responsible for color development in protein samples. Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA).

Reagents: 

BCA Protein Assay kit is available from Pierce, Rockford, Il 61105, USA. The reagent kit has the following reagents:

Reagent A (sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartarate in 0.1 M sodium hydroxide)

Reagent B (4% cupric sulfate)

Albumin Standard Ampules, 2 mg/ml: Prepare bovine serum albumin (BSA) at 2.0 mg/ ml in 0.9% saline and 0.05% sodium azide

Just before use, prepare working BCA reagent by mixing one part of Reagent B with 50 parts of Reagent A. 

Protocol:

Spectrophotometer-

Prepare a series of dilutions of known concentration from the protein and assay alongside the protein sample of unknown concentration.

1. Prepare standard series by taking aliquots of the BSA protein in different tubes so that amount of the protein in different tubes varies from 5 to 50 μg protein.

2. Take aliquot of protein and water to make the volume 1.0 ml.

3. Prepare the blank by taking 1.0 ml of water.  

4. Add 2 ml of working BCA reagent in each tube and shake well.

5. Incubate all the tubes at 37^oC for 30 minutes.

6. Measure the absorbance at 562 nm in a spectrophotometer.

7. Taking into account the readings with standard proteins, plot a calibration curve taking absorbance on the vertical axis and amount of the protein on the horizontal axis.

8. From this plot, using absorbance value with the unknown protein, determine the amount of the protein in the unknown sample.

Plate reader-

1. Prepare standard series by taking aliquots of the BSA protein in different wells so that amount of the protein varies from 0-25 μg protein. 

Standard (Volume in µl)

H2O	25	20	15	10	0
BSA (1 µg/µl)	0	5	10	15	25

2. Take aliquot of protein and water to make the volume 25 µl.

3. Add 200 µl of working BCA reagent in each well and place the plate on shaker at 37 ^oC for 30 minutes in dark.

4. Measure the absorbance at 562 nm using plate reader.

5. Taking into account the readings with standard proteins, plot a calibration curve taking absorbance on the vertical axis and amount of the protein on the horizontal axis.

6. From this plot, using absorbance value with the unknown protein, determine the amount of the protein in the unknown sample.

Although the method has several advantages viz. compatibility with ionic and non-ionic detergents, stable working reagent, less protein to protein variation, broad linear working ranges and good sensitivity; reducing agents and copper chelators do interfere with this assay. This problem can be ruled out by removing the interfering substances prior to BCA analysis by precipitating the protein sample with TCA or acetone and re-dissolving the protein pellet in NaOH or water. Some people prefer to bind proteins to positively charged nylon at alkaline pH and washing out the non-bound interfering agents.