Pierce Chemical Technical Library

assays: protein
Bicinchoninic Acid Reducing agents and copper chelators do interfere
with this assay; however, interfering substances
The BCA protein color reaction can be accelerated
at room temperature with the addition of borate
(BCA) Method can be successfully eliminated prior to BCA analy- ions to the reagent.14 This acceleration reduces the
sis. This has been accomplished by precipitating effect of some substances known to interfere in the
One of the most recent advances in protein assay the protein sample away from the interfering standard and micro-BCA based assays. The faster
technology is the use of bicinchoninic acid (BCA) agents with TCA or cold acetone and redissolving BCA color reaction of this special formulation
in a unique reagent system. BCA is a highly sensi- the protein pellet in an SDS/NaOH solubilizing makes the assay adaptable to the kinetic mode and
tive and selective detection reagent for the cuprous reagent or deionized water. 35-37 An alternative allows it to be run in automated or semi-automated
cation (Cu+1). This protein assay combines the method describes the elimination of interfering systems.
well-known reduction of Cu+2 by protein to Cu+1 in substances such as thiols and reducing sugars
an alkaline medium with the cuprous (Cu+1) ion de- The Pierce BCA Protein Assay Kit has been studied
prior to the BCA assay by binding proteins to posi- independently by Shibuya and coworkers, 41
tecting property of BCA.12 tively charged nylon at alkaline pH and washing out demonstrating the dependence of the assay on dif-
The macromolecular structure of the protein, the the non-bound interfering agents.38 ferent types of buffers and concentrations of alkali
number of peptide bonds and the presence of four A report by Sorensen takes advantage of the inter- in both the standard and enhanced protocol. A BCA
amino acids (cysteine, cystine, tryptophan and ty- ference of ascorbic acid with the BCA Protein modification has been described that allows the
rosine) have been reported to be responsible for Assay to ascertain the quality of a gel filtration de- rapid and reliable analysis of a large number of
color formation in protein samples when assayed salting of a protein solution. The addition of ascor- small volume (5 µl) samples using the commercial
with BCA. Studies with di-, tri- and tetrapeptides bic acid to the protein sample undergoing the de- Pierce BCA kit in a microplate format with a mi-
suggest that the extent of color formation is not salting makes possible the identification of both croplate reader at dual wavelengths.42 Others have
the sum of the contributions of individual color- the protein peak and the salt peak in one step.39-40 taken the commerical kit and reported a modifica-
producing functional groups. Compounds with Using the concept of BCA as a visual monitor of tion that results in sensitive and reproducible pro-
functional groups similar to those of cysteine, cys- protein eluted from a gel filtration column, he has tein determinations in seconds using a standard
tine, tyrosine or tryptophan are known to react also developed a method to locate protein-enzyme microwave oven.43
with BCA.13 conjugates. Because BCA does not interfere with
The purple-colored reaction product of this assay enzyme-substrate reactions, tubes that give both a
is formed by the interaction of two molecules of strong protein and strong enzyme signal indicate
BCA with one cuprous ion (Cu+1). This complex is the presence of the conjugate.40
water soluble and exhibits a strong absorbance at
562 nm, allowing the spectrophotometric quantita-
tion of protein in aqueous solution.
The BCA method eliminates the need for precisely- Figure 4: Proteins react with alkaline copper II to
timed reagent additions and vortexing inherent STEP 1.
0H- produce copper I. BCA then reacts with copper I
with the Lowry method, providing enhanced flexi- Protein + Cu +2 Cu +1
to form an intense purple color at 562 nm.
bility and ease of use. Additional advantages of the
BCA method include: compatibility with ionic and STEP 2.
nonionic detergents; a stable working reagent; less
protein-to-protein variation; broad linear working
ranges with excellent sensitivity; and the flexibility -00C N N COO-
to easily change the protocol.
Cu +BCA*
+1
Cu +1

-00C N BCA* N COO-

Figure 3: BCA M.W. 388.27 Cu+1
Complex

Na0 2 C C0 2 Na

N N

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Internet: http://www.piercenet.com E-mail: [email protected]
Pierce Chemical Technical Library

assays: protein
The Pierce patented BCA Protein Assay products reagent turns purple on reaction with proteins.
deliver fast and reliable results with detergent- Most assays are completed within 20-30 minutes.
compatible chemistry. Three kits are available: the The longer the reaction is allowed to take, the bet-
BCA Protein Assay Kit (Product #23225); the ter the sensitivity. Additional sensitivity can be
Micro-BCA Protein Assay Kit (Product #23235); achieved by incubating at elevated temperatures
and the Kinetic BCA Protein Assay Kit (Product (up to 60°C has been used). Pierce has added bo-
#23250). Should you run low on one component rate to the buffers in its Kinetic BCA* Assay to offer
included with your BCA Protein Assay Kit, replace- further reduction of interference from substances
ment components for the BCA and Micro BCA Kits often found in biological samples such as urine.
are also available The Pierce BCA Protein Assay
products are unique and offer many advantages,
including: Figure 5: Standard Protocol for BCA Protein Assay Reagent.
y g
• Sample may contain detergents without
interference
Add Incubate Read at
• Absolutely linear standard curve Sample 30 min. 37° C 562 nm

• Easily modified sensitivity using time or

temperature
• Low protein-to-protein variability
Each kit consists of two reagents that are mixed
immediately before use and used within 24 hours.
The kit components are stable for at least one year
at room temperature. The apple green working

References
12. 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, 76-85.
13. Wiechelman, K., Braun, R. and Fitzpatrick, J. (1988). Investigation of the bicinchoninic acid protein assay identification of the groups responsible for color
formation. Anal. Biochem. 175, 231-237.
14. Shihabi, Z.K. and Dyers, R.D. (1988). Protein analysis with bicinchoninic acid. Annals Clin. Lab Sci. 18, 235-239.
35. Brown, R.E., Jarvis, K.L. and Hyland, K.J. (1989). Protein measurement using bicinchoninic acid; elimination of interfering substances. Anal. Biochem. 180,
136-139.
36. BCA Application Note #12; Elimination of soluble interfering substances from samples in preparation for protein estimation by Pierce’s BCA* Protein Assay
Reagent (1990). Pierce Chemical Company, Rockford, Il 61105.
37. BCA Application Note #13; Acetone precipitation: elimination of soluble interfering substances from samples in preparation for protein estimation by
Pierce’s BCA* Protein Assay Reagent. Pierce Chemical Company, Rockford, Il 61105.
38. Gates, R.E. (1991). Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay. Anal. Biochem. 196(2), 290-
295.
39. Sorensen, K. (1992). One reagent simultaneously identifies the salt and the protein peaks on desalting column. BioFeedback 12(2).
40. BCA Application Note #11; The Sorensen Method: Use of BCA for determination of protein and reducing reagents. Use of BCA to locate enzyme conjugates.
41. Shibuya, T., Watanabe, Y., Nalley, K.A., Fusco, A. and Salafsky, B. (1989). The BCA protein determination system. An analysis of several buffers, incubation
temperature and protein standards. Tokyo Ika Daigaku Zasshi 47(4), 677-682.
42. Hinson, D.L. and Webber, R.J. (1988). Miniaturization of the BCA Protein Assay. BioTechniques 6(1), 14,16,19.
43. Akins, R.E. and Tuan, R.S. (1992). Measurement of protein in 20 seconds using a microwave BCA assay. BioTechniques 12(4), 496-7, 499.

Telephone: 800.874.3723 or 815.968.0747 Fax: 800.842.5007 or 815.968.7316

Internet: http://www.piercenet.com E-mail: [email protected]