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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN QUANTITATION

SIMPLIFIED BICINCHONINIC ACID (BCA) ASSAY FOR PROTEIN CONCENTRATION DETERMINATION USING MICROTITER PLATES

Simplified Bicinchoninic Acid (BCA) Assay for Protein Concentration Determination Using Microtiter Plates
Contributor: The Laboratory of Bruce N. Ames at the University of California, Berkeley and the Children's Hospital of Oakland Research Institute
 
Overview
The Bicinchoninic Acid (BCA) test to determine protein concentration is a highly sensitive alternate assay to the Lowry assay. It is easier to carry out and sensitivity can be varied using different temperatures. Furthermore, the dye complex is very stable. Unfortunately, this test is highly susceptible to interference. However, its insensitivity to detergents is similar to that of the Lowry method.Using a 96-well microtiter plate makes the BCA assay for protein quantification quicker and easier. In addition, this simplified method requires less sample volume (ten-fold less volume standard) and less BCA reagents.
 
Procedure
1. Prepare a set of protein standards of known concentration (between 0 to 2.5 mg protein/ml).

2. Pipette 10 μl of each standard or sample in duplicate (or triplicate) wells in a microtiter plate. Include blanks lacking protein.

3. Add 200 μl Working Reagent to each well.

4. Mix well by pipetting up and down several times (see Hint #1).

5. Incubate at room temperature for 2 hr (see Hint #2).

6. Determine the absorbance at 562 nm using a spectrophotometric microtiter plate reader.

7. Calculate a linear regression curve (see Hint #3).

8. Calculate concentration of unknown samples using the linear regression curve.

Solutions
Working Reagent   Mix 100 volumes Reagent A with 2 volumes Reagent B
Stable for approximately 1 week
Solution should be clear and green colored.
Reagent B   Prepare in 10 ml of ddH2O
0.4 g Cupric Sulfate, Pentahydrate
10 M NaOH
Reagent A   0.95 g Sodium Bicarbonate (NaHCO3)
2 g Sodium Carbonate (Na2HCO3)
Adjust the pH to 11.25 with 10 M NaOH
Bring to 100 ml with ddH2O
0.16 g Sodium Tartrate
1 g Sodium Bicinchoninate
0.4 g NaOH
Protein Standards   0 to 2.5 mg/ml Bovine Serum Albumin (other protein sources could also be used)
Prepared in a solution that is the same as the protein samples
 
BioReagents and Chemicals
Sodium Carbonate
Sodium Bicarbonate
Bovine Aerum Albumin
Cupric Sulfate
Sodium Bicinchoninate
Sodium Hydroxide
Sodium Tartrate
 
Protocol Hints
1. Do not introduce any bubbles into the solution.

2. The samples can also be incubated at 60°C for 30 min. Cool samples before reading the absorbance. Color is stable for at least 1 hour. The assay shows greater variability among proteins and is less sensitive when it is performed at room temperature.

3. Linear regression analysis: plot along the X-axis the protein standard concentration (mg/ml) and along the Y-axis the absorbance value at 562 nm. Calculate the best linear fit (y = mx + b) where y is mg/ml, m is the slope of the line, b is the y-intercept, and x is the absorbance value.