1. Prepare a standard curve of five to six concentrations of a protein standard, which is representative of the protein solution to be tested. Usually 0 – 10 ug of protein adjusted to 800 uL with water or sample buffer.
  2. Pipet 10 uL each sample into a tube and adjust volume to 800 uL (NOTE: the volume of the unknown can be adjusted to as much as 800 uL). Samples are usually assayed in duplicate or triplicate.
  3. Add 200 uL of the dye reagent (DO NOT DILUTE). Vortex each tube.
  4. Incubate at room temperatre for at least 5 minutes. Absorbance will increase over time; samples should incubate at room temperature for no more than 1 hour.
  5. Measure absorbance at 595 nm