1.2.2. The bicinchoninic acid (BCA) assay
The BCA assay (Smith et al., 1985) uses bicinchoninic acid (BCA) in a reaction forming Cu+ from Cu2+ by the Biuret complex in alkaline solutions of protein. The advantage of this assay is its high tolerance towards the presence of detergents in protein extracts.
- Prepare Reagent A (aqueous solution containing 1 % of 2,2'-Biquinoline-4,4-dicarboxylic acid disodium salt, 0.16 % of sodium tartrate, 0.4 % sodium hydroxide, 2 % of Na2CO3·H2O, 0.95 % NaHCO3) .
- Adjust pH to 11.25.
- Prepare Reagent B (4 % CuSO4·5H2O
in deionized water).
These reagents are stable indefinitely when kept in dark bottles at room temperature.
- Standard working reagent (S-WR) should be prepared weekly or as needed by mixing 50 volumes of Reagent A with 1 volume of Reagent B.
- Prepare a convenient standard curve using a solution 1 mg/ml of bovine serum albumin (BSA fraction V) in either isotonic saline or, in the case of any possibly interfering substance (e.g. sodium dodecyl sulphate, SDS), in a solution containing this particular substance.
- Prepare blanks and triplicates of standards and samples (as described above in step 6 of the Bradford assay).
- Add 20 volumes of S-WR per volume of sample (e.g. add
950 µl S-WR to a
50 µl sample).
- Add 1 ml of S-WR to each.
- Vortex well for a few seconds.
- Incubate the samples for 30 min at 37ºC.
- Cool samples to room temperature.
- Transfer to disposable plastic cuvettes.
- Set spectrophotometer to a wavelength of 562 nm.
- Read standard curve and unknown samples.
- Average the absorbance readings of each of the triplicates and subtract blanks from standards and samples.
- Plot absorbance values of the standard curve samples against their protein concentration (µg/µl).
- Determine the concentration of the unknown samples by linear regression.
- Make sure that the standard curve is linear and that unknown samples are within range.