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.

  1. 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) .
  2. Adjust pH to 11.25.
  3. Prepare Reagent B (4 % CuSO4·5H2O in deionized water).
    These reagents are stable indefinitely when kept in dark bottles at room temperature.
  4. 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.
  5. 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.
  6. Prepare blanks and triplicates of standards and samples (as described above in step 6 of the Bradford assay).
  7. Add 20 volumes of S-WR per volume of sample (e.g. add 950 µl S-WR to a
    50 µl sample).
  8. Add 1 ml of S-WR to each.
  9. Vortex well for a few seconds.
  10. Incubate the samples for 30 min at 37ºC.
  11. Cool samples to room temperature.
  12. Transfer to disposable plastic cuvettes.
  13. Set spectrophotometer to a wavelength of 562 nm.
  14. Read standard curve and unknown samples.
  15. Average the absorbance readings of each of the triplicates and subtract blanks from standards and samples.
  16. Plot absorbance values of the standard curve samples against their protein concentration (µg/µl).
  17. Determine the concentration of the unknown samples by linear regression.
  18. Make sure that the standard curve is linear and that unknown samples are within range.