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:
- 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.
- BCA is more tolerant to the presence of compounds that interfere with the Lowry assay.
- 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:
- 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.
- Reagent B: CuSO4 . 5H2O (0.4 g) in 10 mL of water.
- 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.
- 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.
- Reagent B: BCA (4.0 g) in 100 mL of water.
- Reagent C: CuSO4 . 5H20 (0.4 g) in 10 mL of water.
- 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 (BCA) method are:
- To a 100-1aL aqueous sample containing 10-100 ~g protein, add 2 mL of SWR. Incubate at 60~ for 30 min.
- Cool the sample to room temperature, then measure the absorbance at 562 nm.
- A calibration curve can be constructed using dilutions of a stock 1 mg/mL solution of bovine serum albumin (BSA).
- To 1.0 mL of aqueous protein solution containing 0.5-1.0 ~tg ofprotein/mL, add 1 mL of SWR.
- Incubate at 60 ~ for 1 h.
- Cool, and read the absorbance at 562 nm.
Now you know the Bicinchoninic Acid (BCA) Methods for protein measurement, then you can apply it to your sample.