Navigate this Article


BCA (Bicinchoninic Acid) Protein Assay   

How to cite Favorites 3 Q&A Share your feedback Cited by


The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. The reduction of copper is mainly caused by four amino acid residues including cysteine or cystine, tyrosine, and tryptophan that are present in protein molecules. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences. Compared to the Bradford assay, the BCA assay is more objective since the universal peptide backbone also contributes to color formation. One disadvantage of the BCA assay compared to the Bradford assay is that it is susceptible to interference by some chemicals present in protein samples, including reducing agents (i.e., DTT and beta—mercaptoethanol), copper chelators (i.e., EDTA, EGTA) and buffers with high concentration, which can be avoided by generating diluted samples.

Keywords: Protein concentration, Protein measument, BSA standards, Bradford assay, Reduction of copper

Materials and Reagents

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


  1. Spectrophotometer (Tecan)


  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.


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.


  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.
Please login or register for free to view full text
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). BCA (Bicinchoninic Acid) Protein Assay. Bio-101: e44. DOI: 10.21769/BioProtoc.44.

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Islam Ali
University of Hong Kong
Hi Fanglian, I am doing experiments using bacterial biofilm samples, I want to use the BCA protein assay kit to quantify the protein content. Can it be used in my experiments? any procedures are required for my sample before being tested with assay kit?
1/16/2018 9:39:05 PM Reply
Akhtar Ali
School of Medicine GNU South Korea
How Can we use The tecan infinite m200 . Any User Manual
4/9/2015 6:48:53 PM Reply
daniel wasswa
london metropolitan university

hi fanglian , i am under taking a research project which involves the evaluation of protein membrane vesicles in erythrocytes , would the same principle apply to my blood sample as in i have to make a several dilution factors ??

11/16/2015 6:20:57 AM

María Teresa Fernández
necesito determinar cuanto de proteína tengo en mi muestra;
cuanta muestra necesito para poder utilizar este método o como se que tanto de mi muestra utilizar en este método?
1/12/2015 10:39:25 PM Reply
Fanglian He

Hi Maria,

Would you lease put your question in English? Thanks.

1/20/2015 1:42:56 PM

María Teresa Fernández

I need to determine how much protein I have in my sample;
How much sample need to use this method or as both of my sample used in this method?

I'm not good in english sorry, but is important for me know that both of my sample can process this method if I have only 1mg can used this method.

I explain?

1/24/2015 5:55:54 AM

Fanglian He

Hi, Maria,

My apologies for the late response. To determine how much sample is used for BCA protein assay, you should first make several dilutions of your sample, like 1:10, 1:20, 1:50... (if your sample is limited, you can start with a higher dilution. The absorbance readings (at 562 nm) which fall into the linear section of the standard curve can be used to calculate the concentration of your undiluted protein sample.

Hope my answer helps. Please let me know if anything is unclear to you.

Good luck,

3/7/2015 12:18:17 AM

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.