BCA (Bicinchoninic Acid) Protein Assay   

Abstract

Materials and Reagents

1. Bovine serum abumin (BSA) (Sigma)
   
2. BCA protein assay reagents (Pierce, catalog number: 23227 )
   
3. BCA working reagent (WR)
   

Equipment

1. Spectrophotometer (Tecan)

Procedure

1. Prepare bovine serum abumin (BSA) standards. Prepare 1 ml of BSA stock (2 mg ml^-1, dissolved in H₂O) and then make serial (5-8) dilutions with a range of 20-2,000 μg ml^-1.
   
   
2. Prepare BCA working reagent (WR). Calculate the total volume of WR needed. Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50: 1, Reagent A: B) (the mixture appears to be clear and green solution).
   
   
3. For test-tube measurement, 2.0 ml of the WR is required for each sample. Sample to WR ratio is 1: 20.
   1. Pipette 0.1 ml of each standard and unknown protein sample replicate into an appropriately labeled test tube. Set two blank tubes in duplicate. For a standard curve, add 0.1 ml H₂O instead of BSA solution. For the protein samples, add 0.1 ml protein preparation buffer.
      
   2. Add 2.0 ml of the WR to each tube and mix well.
      
   3. Cover and incubate tubes at 37 °C for 30 min.
      
   4. Keep all tubes at RT for 10 min before measurement.
      
   5. Take absorbance readings at 562 nm.
      
   
   
4. For microplate measurement, 200 μl of WR reagent is required. Sample to WR ratio is 1: 8 or 1: 20 (when sample is limited).
   1. Pipette 25 or 10 μl of each standard or protein sample replicate into a microplate well. Use water or protein sample preparation buffer as blank solutions for standard curve and protein samples, respectively.
      
   2. Add 200 μl of the WR to each well.
      
   3. Cover and incubate tubes at 37 °C for 30 min.
      
   4. Keep all tubes at RT for 10 min before measurement.
      
   5. Take absorbance readings at 562 nm on a plate reader.
      

Acknowledgments

This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.

References

1. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76-85.
   
2. Instructions for BCA Protein Assay Kit from Thermo Scientific.