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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.

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[Bio101] BCA (Bicinchoninic Acid) Protein Assay

Biochemistry > Protein > Quantification
Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fhe@bio-protocol.org
Bio-protocol author page: a9
3/5/2011, 28566 views, 3 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.44

[Abstract] 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)

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.

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.


How to cite this protocol: He, F. (2011). BCA (Bicinchoninic Acid) Protein Assay. Bio-protocol Bio101: e44. DOI: 10.21769/BioProtoc.44; Full Text



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4/9/2015 6:48:53 PM  

Akhtar Ali
School of Medicine GNU South Korea

How Can we use The tecan infinite m200 . Any User Manual

11/16/2015 6:20:57 AM  

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 ??

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1/12/2015 10:39:25 PM  

María Teresa Fernández
Unach

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?

1/20/2015 1:42:56 PM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

Hi Maria,

Would you lease put your question in English? Thanks.

1/24/2015 5:55:54 AM  

María Teresa Fernández
Unach

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?

3/7/2015 12:18:17 AM  

Fanglian He (Author)
Department of Biology,University of Pennsylvania

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,
Fanglian

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4/17/2011 2:16:48 PM  

Anonymous Lin
Test institute

WR preparation

5/3/2011 1:55:09 PM  

bio-protocol

I am not sure I understand your question. If it was about how to prepare WR solution, it has been described in step 2 of the procedure, mixing BCA Reagent A with Reagent B (Pierce) with a ratio of 50 to 1 (i.e., 10 ml Reagent A with 200 ul Reagent B).

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