Bicinchoninic Acid (BCA) Method: A Protein Assay

The Bicinchoninic acid (BCA) assay first was described by Smith, et al. BCA assay is similar to Lowry assay since it also depends on the conversion of Cu(2+) to Cu(+) under alkaline conditions. The Cu(+) is then detected by reaction with BCA. The reaction results in the development of an intense purple color with an absorbance maximum at 562 nm. BCA method and Lowry are of similar sensitivity, but BCA method is more advantageous compared to Lowry in a few things, here are the advantages:

1. BCA is stable under alkali conditions, so it can be carried out as a one-step process compared to the two steps needed in the Lowry assay.
2. BCA is more tolerant to the presence of compounds that interfere with the Lowry assay.
3. it is not affected by a range of detergents and denaturing agents such as urea and guanidinium chloride, although it is more sensitive to the presence of reducing sugars.

Materials that you need:

Standard Assay

1. Reagent A: sodium bicinchoninate (0.1 g), Na2CO 3.H2O (2.0 g), sodium tartrate (dihydrate) (0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary, adjust the pH to 11.25 with NaHCO3 or NaOH.
2. Reagent B: CuSO4 . 5H2O (0.4 g) in 10 mL of water.
Reagents A and B are stable indefinitely at room temperature. They may be purchased ready prepared from Pierce, Rockford, IL.
3. Standard working reagent (SWR): Mix 100 vol of regent A with 2 vol of reagent B. The solution is apple green in color and is stable at room temperature for 1 week.

Microassay

1. Reagent A: Na2CO3 . H2O (0.8 g), NaOH (1.6 g), sodium tartrate (dihydrate) (1.6 g), made up to 100 mL with water, and adjusted to pH 11.25 with 10M NaOH.
2. Reagent B: BCA (4.0 g) in 100 mL of water.
3. Reagent C: CuSO4 . 5H20 (0.4 g) in 10 mL of water.
4. Standard working reagent (SWR): Mix 1 vol of reagent C with 25 vol of reagent B, then add 26 vol of reagent A.

The Bicinchoninic Acid method are:

Standard Assay

1. To a 100-1aL aqueous sample containing 10-100 ~g protein, add 2 mL of SWR. Incubate at 60 Celcius degree for 30 min.
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The sensitivity of the assay can be increased by incubating the samples longer. Alternatively, if the color is becoming too dark, heating can be stopped earlier. Take care to treat standard samples similarly.
2. Cool the sample to room temperature, then measure the absorbance at 562 nm.
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Following the heating step, the color developed is stable for at least 1 hour.
3. A calibration curve can be constructed using dilutions of a stock 1 mg/mL solution of bovine serum albumin (BSA).
Note, that like the Lowry assay, response to the BCA assay is dependent on the amino acid composition of the protein, and therefore an absolute concentration of protein cannot be determined. The BSA standard curve can only therefore be used to compare the relative protein concentration of similar protein solutions.

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Microassay

1. To 1.0 mL of aqueous protein solution containing 0.5-1.0 ~tg ofprotein/mL, add 1 mL of SWR.
2. Incubate at 60 Celcius degree for 1 h.
3. Cool, and read the absorbance at 562 nm.

Regards

Reference: Number 8 in References