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BCA Protein Assay (Pierce)

Standard Assay

Sensitive between 20 and 1200ug/mL

  1. Prepare the working reagent by mixing 50 parts reagent A with 1 part reagent B. Working reagent is stable for 1 week at room temperature.
  2. Protein sample or standard is made up to 50uL in appropriate buffer, in microfuge tubes. Typical standards are 0, 25, 50, 100, 150, 200 ug BSA. Often, two dilutions of sample are tested.
  3. Add 1mL working reagent to each tube, mixing well without foaming.
  4. Incubate at 37 C for 20 to 30 min.
  5. Measure absorbance at 562 nm wavelength (visible). Plot standard curve, A vs. ug standard, read unknowns.

To measure amounts between 5 and 250ug, incubate at 60 C for 30 minutes, read as above. Assay volume can be adjusted for 120uL proportionally.

High detergent concentrations and high pH do not interfere with this assay.

Tris buffers above 0.1M and glycine buffers, must be adjusted above pH 11. EDTA above 10mM and other strong chelators can interfere.

Assay is a modified Lowry assay, with bicinchonic acid as chelator or cuprous ions.