Protein Assay by Bradford Method
- Lyophilized bovine plasma gamma globulin or bovine serum albumin (BSA)
- Coomassie Brilliant Blue
- 0.15 M NaCl
- Spectrophotometer and tubes
- Micropipettes
Procedure (Standard Assay, 20–150 µg protein; 200–1500 µg/mL)
- Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750, and 1500 µg BSA/µL. Also prepare serial dilutions of the unknown sample to be measured.
- Add 100 mL of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
- Add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion.
- Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube from step 3, which contains 0 BSA.
- Wait 5 minutes and read each of the standards and each of the samples at a 595-nm wavelength.
- Plot the absorbance of the standards versus their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
Procedure (Micro Assay, 1–10 µg protein)
- Prepare standard concentrations of BSA of 1, 5, 7.5, and 10 µg/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions.
- Add 100 µL of each of the above to separate tubes (use microcentrifuge tubes). Add 1.0 mL of Coomassie Blue to each tube.
- Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5-mL cuvettes.
- Wait 2 minutes and read the absorbance of each standard and sample at 595 nm.
- Plot the absorbance of the standards versus their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.