USER GUIDE

Pierce™ BCA Protein Assay Kit

Catalog Numbers 23225 and 23227
Doc. Part No. 2161296 Pub. No. MAN0011430 Rev. B.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and
gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Introduction ™ ™
The Thermo Scientific Pierce BCA Protein Assay Kit is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric
detection and quantitation of total protein. This method combines the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the
biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing
bicinchoninic acid (see reference 1 on page 4). The purple-colored reaction product of this assay is formed by the chelation of two molecules of
BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein
concentrations over a broad working range (20–2000 µg/mL). The BCA method is not a true end-point method; that is, the final color continues to
develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be
assayed together.
The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine,
tryptophan and tyrosine) are reported to be responsible for color formation with BCA (see reference 2 on page 4). Studies with di-, tri- and
tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups (see
reference 2 on page 4). Accordingly, protein concentrations generally are determined and reported with reference to standards of a common
protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the
unknowns before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is
required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG)
standard (see “Related products“ on page 3) may be used when assaying immunoglobulin samples.
Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1 mL) of protein sample; however, because it
uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized. The Microplate Procedure affords the sample
handling ease of a microplate and requires a smaller volume (10–25 µL) of protein sample; however, because the sample to working reagent ratio is
1:8 (v/v), it offers less flexibility in overcoming interfering substance concentrations and obtaining low levels of detection.
™ ™ ™
Note: For peptide sample concentration measurements, use the Thermo Scientific Pierce Quantitative Fluorometric Peptide Assay or the Pierce
™
Quantitative Colorimetric Peptide Assay Kit (see Related Thermo Scientific Products).

Preparation of standards and working reagent (required for both assay procedures)
Preparation of diluted albumin (BSA) standards
Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the samples.
Use the following table as a guide to prepare a set of protein standards. Each 1 mL ampule of 2 mg/mL Albumin Standard is sufficient to prepare a
set of diluted standards for either working range suggested in the table. There will be sufficient volume for three replications of each diluted
standard.
Table 1 Preparation of diluted albumin (BSA) standards
Dilution Scheme for Standard Test Tube Protocol and Microplate Procedure (Working Range = 20–2,000 µg/mL)
Vial Volume of Diluent (µL) Volume and Source of BSA (µL) Final BSA Concentration (µg/mL)
A 0 300 of Stock 2000
B 125 375 of Stock 1500
C 325 325 of Stock 1000
D 175 175 of vial B dilution 750
E 325 325 of vial C dilution 500
F 325 325 of vial E dilution 250
G 325 325 of vial F dilution 125
H 400 100 of vial G dilution 25
I 400 0 0 = Blank
Dilution Scheme for Enhanced Test Tube Protocol (Working Range = 5–250 µg/mL)
Vial Volume of Diluent (µL) Volume and Source of BSA (µL) Final BSA Concentration (µg/mL)
A 700 100 of Stock 250
B 400 400 of vial A dilution 125
C 450 300 of vial B dilution 50
D 400 400 of vial C dilution 25
E 400 100 of vial D dilution 5
F 400 0 0 = Blank
For Research Use Only. Not for use in diagnostic procedures.
30 January 2020
Preparation of the BCA working reagent (WR)
1. Use the following formula to determine the total volume of WR required:
(# standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required
Example: for the standard test-tube procedure with 3 unknowns and 2 replicates of each sample:
(9 standards + 3 unknowns) × (2 replicates) × (2 mL) = 48 mL WR required
Note: 2.0 mL of the WR is required for each sample in the test-tube procedure, while only 200 µl of WR reagent is required for each sample in
the microplate procedure.
2. Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). For the above example, combine 50 mL of
Reagent A with 1mL of Reagent B.
Note: When Reagent B is first added to Reagent A, turbidity is observed that quickly disappears upon mixing to yield a clear, green WR.
Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for several days when stored in a closed
container at room temperature (RT).

Procedure summary (test-tube procedure, standard protocol)

Test-tube procedure (sample to WR ratio = 1:20)

1. Pipette 0.1 mL of each standard and unknown sample replicate into an appropriately labeled test tube.
2. Add 2.0 mL of the WR to each tube and mix well.
3. Cover and incubate tubes at selected temperature and time:
• Standard Protocol: 37°C for 30 minutes (working range = 20–2000 µg/mL)
• RT Protocol: RT for 2 hours (working range = 20–2000 µg/mL)
• Enhanced Protocol: 60°C for 30 minutes (working range = 5–250 µg/mL)
Note:
· Increasing the incubation time or temperature increases the net 562 nm absorbance for each test and decreases both the minimum detection
level of the reagent and the working range of the protocol.
· Use a water bath to heat tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Using a forced-air incubator
can introduce significant error in color development because of uneven heat transfer.
4. Cool all tubes to RT.
5. With the spectrophotometer set to 562 nm, zero the instrument on a cuvette filled only with water, then measure the absorbance of all the
samples within 10 minutes.
Note: Because the BCA assay 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, no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made
within 10 minutes of each other.
6. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562nm absorbance measurement of all other
individual standard and unknown sample replicates.
7. Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL.
Use the standard curve to determine the protein concentration of each unknown sample.

Microplate procedure (sample to WR ratio = 1:8)

1. Pipette 25 µL of each standard or unknown sample replicate into a microplate well (working range = 20–2000 µg/mL). (For example,Thermo
™ ™
Scientific Pierce 96–Well Plates, Product No. 15041).
Note: If sample size is limited, 10 µL of each unknown sample and standard can be used (sample to WR ratio = 1:20). However, the working
range of the assay in this case is be limited to 125–2000 µg/mL.
2. Add 200 µL of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.
3. Cover plate and incubate at 37°C for 30 minutes.
4. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.
Note:
· Wavelengths from 540–590 nm have been used successfully with this method.
· Because plate readers use a shorter light path length than cuvette spectrophotometers, the Microplate Procedure requires a greater sample to
WR ratio to obtain the same sensitivity as the standard Test Tube Procedure. If higher 562 nm measurements are desired, increase the
incubation time to 2 hours.
· Increasing the incubation time or ratio of sample volume to WR increases the net 562 nm measurement for each well and lowers both the
minimum detection level of the reagent and the working range of the assay. As long as all standards and unknowns are treated identically,
such modifications may be useful.

2 Pierce™ BCA Protein Assay Kit User Guide

 5. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm measurements of all other individual
standard and unknown sample replicates.
6. Prepare a standard curve by plotting the average Blank–corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL.
Use the standard curve to determine the protein concentration of each unknown sample.
Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best–fit curve provides more
accurate results than a purely linear fit. If plotting results by hand, a point–to–point curve is preferable to a linear fit to the standard points.

Related products
Cat. NO. Product
15041 Pierce™ 96-Well Plates, 100/pkg.
15075 Reagent Reservoirs, 200/pkg.
15036 Sealing Tape for 96-Well Plates, 100/pkg.
23209 Albumin Standard Ampules, 2mg/mL, 10 × 1mL ampules, containing bovine serum albumin (BSA)
23208 Pre-Diluted Protein Assay Standards: Bovine Serum Albumin (BSA) Set, 7 × 3.5mL
23212 Bovine Gamma Globulin Standard, 2mg/mL, 10 × 1mL ampules
23213 Pre-Diluted Protein Assay Standards, (BGG) Set, 7 × 3.5mL aliquots
23246 Pierce™ Detergent Compatible (Bradford)™ Assay Kit
23235 Pierce™ Micro BCA™ Protein Assay Kit
23290 Pierce™ Quantitative Fluorometric Peptide Assay
23275 Pierce™ Quantitative Colorimetric Peptide Assay
23236 Coomassie Plus™ (Bradford)™ Assay Kit
23215 Compat-Able™ Protein Assay Preparation Reagent Set
23250 Pierce™ BCA Protein Assay Kit-Reducing Agent Compatible

Additional information
A. Please visit our website for additional information including the following items:
• Tech Tip: Eliminate interfering substances from samples for BCA Protein Assay
B. Alternative Total Protein Assay Reagents
™
If interference by a reducing substance or metal-chelating substance contained in the sample cannot be overcome, try the Thermo Scientific
™ ™
Coomassie Plus (Bradford) Assay Kit (Product No. 23236), which is less sensitive to such substances.
C. Cleaning and Re-using Glassware
Exercise care when re-using glassware. All glassware must be cleaned and given a thorough final rinse with ultrapure water.
D. Response characteristics for different proteins
Each of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences
relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s
color response. Most protein assay methods use BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the
sample is determined (Figure 1). However, if great accuracy is required, prepare the standard curve from a pure sample of the target protein.
BCA Protein Assay
3
BGG

BSA
Net A(562 nm)

0
0 500 1,000 1,500 2,000
Protein Concentration in µg/mL

Fig. 1 Typical color response curves for BSA and BGG using the standard test tube protocol (37°C/30‑minute incubation).
Typical protein-to-protein variation in color response is listed in Table 2. All proteins were tested at 1000 µg/mL using the 30-minute/37°C Test Tube
Protocol. The average net color response for BSA was normalized to 1.00 and the average net color response of the other proteins is expressed as a
ratio to the response of BSA.

Pierce™ BCA Protein Assay Kit User Guide 3

Table 2 Protein-to-protein variation. Absorbance ratios (562 nm) for proteins relative to BSA using the standard test tube protocol.
Ratio = (Avg “test” net Abs.) / (avg. BSA net Abs.)
Protein Tested Ratio
Albumin, bovine serum 1.00
Aldolase, rabbit muscle 0.85
α-Chymotrypsinogen, bovine 1.14
Cytochrome C, horse heart 0.83
Gamma globulin, bovine 1.11
IgG, bovine 1.21
IgG, human 1.09
IgG, mouse 1.18
IgG, rabbit 1.12
IgG, sheep 1.17
Insulin, bovine pancreas 1.08
Myoglobin, horse heart 0.74
Ovalbumin 0.93
Transferrin, human 0.89
Average Ratio 1.02
Standard Deviation 0.15
Coefficient of Variation 14.7%

Cited references
1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85.
2. Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation.
Anal Biochem 175:231-7.
3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal Biochem 159:138-42.
4. Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136-9.

Product references
Adilakshami, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for
survival or death. J Biol Chem 277:4147-51.
Fischer, T., et al. (1999). Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro. Proc Nat Acad Sci 96:6722-7.
Prozialeck, W.C., et al. (2002). Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequester β-catenin in human cervical
epithelial cells. Infection and Immunity 70:2605-13.
Roberts, K.P., et al. (2002). A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D
and E. Biology of Reproduction 67:525-33.

Troubleshooting and FAQs

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database is frequently updated to ensure accurate and thorough content.
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Revision history: Pub. No. MAN0011430
Revision Date Description
B.0 30 January 2020 Moved troubleshooting content to thermofisher.com.
A.0 17 October 2015 Baseline for revision.

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For Research Use Only. Not for use in diagnostic procedures.

30 January 2020