INSTRUCTION MANUAL

BCA Protein Micro Assay Kit

Kit for Protein Qantification

(Cat. No. 39229)

SERVA Electrophoresis GmbH  Carl-Benz-Str. 7  D-69115 Heidelberg

Phone +49-6221-138400, Fax +49-6221-1384010
e-mail: [email protected]  http://www.serva.de
Contents

1. BCA PROTEIN MICRO ASSAY KIT 2

1.1. General information 2
1.2. Kit components 2
1.3. Additionally required equipment 2
1.4. Storage conditions 3

2. PROCEDURE OF THE BCA ASSAY 3

2.1. Preparation of standards and reagents 3
2.1.1. BSA Protein standard 3
2.1.2. BCA working reagent (BWR) 3
2.2. Preparation of BSA reference solutions 3
2.3. Protein quantification procedure 4
2.3.1. Protocol 4
2.4. Calculation of protein concentrations 5
2.4.1. Creation of the calibration curve 5
2.4.2. Calculation of the protein concentration 6

3. LITERATURE 7

4. TROUBLESHOOTING 8

Vers. 07/10

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1. BCA Protein Micro Assay Kit
1.1. General information

The bicinchoninic acid (BCA) based protein assay is a most sensitive and detergent
compatible method for the colorimetric detection and quantitation of total protein. This
method is a combination of the well-known Biuret reaction, the reduction of Cu2+ to
Cu1+ by protein in an alkaline medium, and the highly sensitive and selective
colorimetric detection of the cuprous cation (Cu1+) with a bicinchoninic acid
containing reagent. The purple-colored reaction product of this assay is formed by
the chelation of two molecules of BCA with one Cu1+ ion. This water-soluble complex
shows a strong absorbance at 562 nm.

BCA-Protein Reaction

1. Protein (peptide bonds) + Cu2+ • Cu1+ Biuret complex

2. Cu1+ + 2 Bicinchoninic Acid • BCA-Cu1+ complex (purple colored, read at

562 nm).

The SERVA BCA Protein Micro Assay kit contains well standardized reagents which
give a linear increase in the reaction product at 562 nm with increasing protein
concentrations over a broad range from 0.5 µg/ml to 20 µg/ml. The macromolecular
protein structure, the number of peptide bonds and the presence of four amino acids
(cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color
formation with BCA.

1.2. Kit components

Component
Reagent A: Buffer Solution 125 ml
Reagent B: BCA Solution 125 ml
Reagent C: Copper Sulfate Solution 10 ml
Protein Standard (Bovine Serum Albumine) BSA 10 x 2 mg

1.3. Additionally required equipment

• Vortex mixer

• Photometer suitable for measurement at 562 nm wavelength and the usage of

microcuvettes

• Plastic cuvettes

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1.4. Storage conditions
The recommended storage temperature for the BCA Protein Micro Assay Kit is
+2 – +8 °C. Under these storage conditions the unopened reagent is at least useable
until: see expiry date on the label.

2. Procedure of the BCA Assay

2.1. Preparation of standards and reagents

2.1.1. BSA Protein standard
Tap the powder in the standard vial to the bottom and add 1 ml of water containing
0.05 – 0.1 % sodium azide. The BSA stock solution (2.0 mg/ml) can be stored at
4 ºC for two weeks.
Prepare a fresh set of protein standards by diluting the BSA stock solution,
preferably in the same diluents as your sample. Please refer to 2.2. for details about
dilution of the BSA stock solution. One vial of BSA Standard is sufficient to prepare
three sets of diluted standards.

2.1.2. BCA working reagent (BWR)

To prepare BWR, mix 25 parts of Reagent A with 25 parts of Reagent B and 1 part of
Reagent C. Prepare sufficient volume of BWR based upon the number of tests to be
done. Each test tube sample to be done requires 0.5 ml of the BWR.

2.2. Preparation of BSA reference solutions

Final BSA Volume BSA Volume

Concentration Diluent
[µg/ml] [µl] [µl]
20 (A) 100 (Stock solution) 9900
15 (B) 1500 (A) 500
10 (C) 1000 (A) 1000
5 (D) 500 (A) 1500
2,5 (E) 250(A) 1750
1 (F) 100 (A) 1900
0,5 (G) 50 (A) 1950

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2.3. Protein quantification procedure

Each of the commonly used total protein assay methods exhibit some degree of
varying response toward different proteins. These differences relate to amino acid
sequence, pI, protein structure and the presence of certain side chains or prosthetic
groups that can dramatically alter the protein’s color response. Most protein assay
methods utilize BSA or immunoglobulin G (IgG) as standard against which the
concentration of protein in the sample is determined.

The following substances have been reported to interfere with the accurate
estimation of protein concentration with the BCA Reagent. They should be avoided
as components of the sample buffer.

Ascorbic Acid Catecholamin Creatinine

Cysteine EGTA Impure Glycerol
Hydrogen peroxide Hydrazides Iron
Lipids Melibiose Phenol Red
Impure Sucrose Tryptophan Tyrosine
Uric Acid

2.3.1. Protocol
• Pipet 500 µl of each standard or unknown sample into appropriately labeled
test tubes. Use 500 µl of the diluent for the blank tubes.
• Add 500 µl of the BWR to each tube, mix well.
• Incubate all the tubes at the selected temperature and time:
Standard Protocol: 37 °C for 30 min.
Enhanced Protocol: 60 °C for 15 min.
• After incubation, cool all tubes to RT.
• Measure the absorbance at 562 nm (A562nm) of each tube vs. a water
reference.
Note: Because the BCA Reagent does not reach a true end point, color
development will continue even after cooling to RT. However, because the rate of
color development is low at RT, if the A562nm readings of all the tubes can be done
in 10 minutes or less, no significant error is introduced.
• Subtract the average A562nm reading of the blanks from the A562nm reading for
each standard or unknown sample.
• Prepare a standard curve by plotting the average blank corrected A562nm
reading for each BSA standard vs. its concentration in µg/ml. Using the
standard curve, determine the protein concentration for each unknown
sample.

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 Flowchart:
Pipet 500 µl dist. H2O and buffer for blank, reference
solutions and samples into test tubes

Add 500 µl BCA working reagent

Mix by vortexing

Incubate according to the standard or modified

protocol

Let the tubes cool down to RT

Transfer solutions to cuvettes

Measure absorbance at 562 nm (A562nm)

2.4. Calculation of protein concentrations

2.4.1. Creation of the calibration curve

Create a table with the absorbance results obtained from the assay. From the values
obtained for the BSA reference solutions create a calibration curve which is used to
determine the protein concentration in the unknown sample.

Figure 1: Example of BSA calibration curve (incubation 30 min at 37 °C)

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2.4.2. Calculation of the protein concentration
• Construct a calibration curve by plotting optical density reading on y-axis
against standard protein amount (µg/tube) on the x-axis
• Record the value x for the protein amount per tube from the calibration curve
corresponding to the optical density reading for inividual sample.
• Calculate the sample concentration (C) by using the following formula:

X [µg] / V [µl] = C [µg/µl] = C [mg/ml]

X: protein amount of the sample

V: sample volume used in the assay

• Correct for predilution of samples if any by using the following formula:

(X [µg] / V [µl]) * DF = C [µg/µl] = C [mg/ml]

X: protein amount of the sample

V: sample volume used in the assay
DF: dilution factor

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3. Literature
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.
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.
Brown, R., Jarvis, K. and Hyland, K. (1989). Protein measurement using
bicinchoninic acid: elimination of interfering substances. Anal. Biochem.
Peterson, G.L. (1979) . Review of the folin phenol protein quantitation method of
Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100, 201-220.
Kirschbaum, G. (1986). Use of the bicinchoninic acid assay in measuring urinary
proteins. Clin. Chem. 32, No. 3, Letter to the Editor, 572.

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4. Troubleshooting
• The presence of incompatible substances in the sample
Interference in the BCA Protein Assay may be eliminated or overcome by:
a. Removing the interfering substance by using the SERVA BluePrep kits, e.g.
DetergentEx, CBD, as well as dialysis or gel filtration.
b. Diluting the sample to the point that the substance no longer interferes. (Can
be done if the starting protein concentration of the sample is high.)
c. Precipitating the proteins in the sample with acetone or trichloroacetic acid
(TCA); the liquid containing the interfering substances is discarded and the
protein pellet is easily solubilized directly in the BWR.
d. Increasing the amount of copper in the BWR may eliminate interference by
copper chelating agents.
Note: For higher accuracy of the estimate of protein concentration in the sample, the
protein standards must be treated identically to the sample.
• Reading at wavelengths other than 562 nm
If a photometer or plate reader with a 562 nm filter is not available, the purple
color may be measured at any wavelength between 540 nm and 590 nm. The
maximum absorbance of the BCA-Cu1+ complex occurs at 562 nm. Taking the
absorbance measurements at any wavelength other than 562 nm will result in
a lower slope for the standard curve and may increase the minimum detection
level for the protocol.

• Cleaning and re-use of glassware

Care must be exercised when re-using glassware. The BCA Reagent is
sensitive to metal ions, especially copper ions. All glassware must be cleaned
and then given a thorough final rinse with high-quality deionized water.