Bicinchoninic acid Protein Assay Micro

Kit for Protein Quantitation and Determination in Micro Scale

Product code A7845
The Bicinchoninic acid Protein Assay Micro Kit for protein determination in micro scale is formulated for the
colorimetric detection and quantitation of total protein in dilute samples (0.5-20 g/ml).
Like the Lowry method, the assay relies on the reduction of Cu2+ ions by protein. The Cu+ thus formed is
detected by conversion into a violet-colored substance by reaction with bicinchoninate.
The absorbance at 562 nm of the Cu(I)-(bicinchoninate)2 complex is directly proportional to protein
concentration. The bicinchoninic acid protein micro assay is more sensitive than the Lowry method and less
subject to interference. In particular, it is insensitive to detergents such as Triton X-100 and SDS (1 %).

Important Notes

Precipitates which may form in Reagent A or B during storage should be dissolved by gentle warming and
stirring.

Color development continues even at room temperature. However no significant error is introduced if all
absorbance measurements are completed within 10 minutes.

Reagents that chelate metal ions, change the pH of the assay or reduce copper, are known to nterfere
with the assay. Please check that the following components are not in the sample buffer: ascorbic acid,
catecholamines, creatinine, cysteine, EGTA, impure glycerol, hydrogen peroxide, hydrazides, iron, lipids,
melibiose, phenol red, impure sucrose, tryptophan, tyrosine, uric acid.

Other substances affect the assay to a lesser extent and, if their concentration in the sample buffer is
below a certain value, can be tolerated. Please refer to table 2 for maximum compatible concentrations of
many of these substances.

It is necessary to create a standard curve during each assay regardless of the format used. The
Bicinchoninic acid Protein Assay Micro working solution is stable for several days at RT. If not used
immediately, it should be stored at RT in a closed container.

Required Equipment/Materials

Spectrophotometer capable of measuring absorbance in the region of 560 nm

Water bath
Test tubes or 96 well plate
Protein Standards
Bicinchoninic acid Protein Assay Micro

A7845,0480

A7845,SAMPLE

Reagent A (tartrate in alkaline carbonate

buffer)

A7845,0480A

240 ml

A7845,SAMPLEA

25 ml

Reagent B (4 % Bicinchoninic acid in H2O)

A7845,0480B

240 ml

A7845,SAMPLEB

25 ml

Reagent C (4 % CuSO4 5H2O in H2O)

A7845,0480C

12 ml

A7845,SAMPLEC

5 ml

Storage:
Shipment:
Stability (Reagents A - C):
Stability (working solution):
Sensitivity:
No. of tests

RT
ambient temperature
2 years
several days
0.5 - 20 g/ml protein
480 test tubes or 3,200 microplate assays

Procedures
Preparation of the Working Reagent
Mix 25 parts of Reagent A, 24 parts of Reagent B and 1 part of Reagent C. The amount of Working Reagent
required for each sample is 1 ml for the Test Tube Procedure and 150 l for the Micro assay Plate Procedure.
A precipitate is initially formed when reagent C (CuSO4) is added to the mixture of Reagents A and B. It will
then redissolve to yield a clear, apple green Working Reagent.
Prepare sufficient volume of Working Reagent for the samples to be assayed plus the calibration standards.
For example, for the standard test tube procedure with 9 standards (including a blank), 3 unknowns and 2
replicates for each sample 24 ml of Working reagent is required. The Working Solution is stable for several
days when stored at room temperature in a closed container.

Preparation of the Calibration Standards

Prepare a fresh set of protein standards in the 0.5-200 g/mL range, preferably using the same diluent as
your sample. While the most accurate results are obtained using a pure sample of the protein to be measured
as standard, in many cases this is expensive or not available. Therefore, the standards are generally prepared
from a 1.0-2.0 mg/mL stock solution of bovine serum albumin. For several proteins a correction factor relative
to BSA is reported in the following table:
Protein

Correction Factor

Protein

Correction Factor

Albumin, bovine serum

1.00

IgG, mouse

1.23

Aldolase, rabbit muscle

0.80

IgG, rabbit

1.12

-Chymotrypsinogen, bovine

0.99

IgG, sheep

1.14

Cytochrome c, horse heart

1.11

Insulin, bovine pancreas

1.22

-globulin, bovine

0.95

Myoglobin, horse heart

0.92

IgG, bovine

1.12

Ovalbumin

1.08

IgG, human

1.03

Transferrin, human

0.98

A. Test tube Procedure

linear working range: 0.5 - 20 g/ml
1.

Pipette 1.0 ml of each standard (including a blank) or unknown is pipetted into a labelled test tube.

2.

Add 1.0 ml of Working Reagent to each tube and thoroughly mix.

3.

Cover the tubes and incubate at 60C in a water bath for 1 hour.

4.

Cool all tubes to RT.

5.

Measure the absorbance at 562 of all samples within 10 minutes.

6.

Subtract the 562 nm absorbance value of the blank from the readings of the standards and the
unknowns.

7.

Plot the blank-corrected 562 nm reading for each standard vs. its concentration.

8.

Determine the protein concentration of each unknown from the calibration plot.

B. Microplate Procedure
linear working range: 2 - 40 g/mL
1.
2.

Pipette a 150 l of each standard (including a blank) or unknown into a microwell plate.
Add 150 l of Working Reagent to each well and mix thoroughly on a plate shaker for 30 sec.

3.

Cover plate and incubate at 37C for 2 hours. Do not incubate at higher temperatures.

4.

Cool plate at room temperature.

5.

Measure the absorbance at 562 of all the samples on a plate reader.

6.

Subtract the 562 nm absorbance value of the blank from the readings of the standards and the
unknowns.

7.

Plot the blank-corrected 562 nm reading for each standard vs. its concentration. Determine the protein
concentration of each unknown from the calibration plot.

Compatibility of Reagents with the Standard

Test Tube Protocol
Salts & Buffers

Reducing & Thiol Containing Agents

Concentration

ACES, pH 7.8

Concentration

10 mM

N-Acetylglucosamine in PBS

NC

Ammonium sulfate

NC

Ascorbic acid

NC

Bicine, pH 8.4

2 mM

Cysteine

NC

Bis-Tris, pH 6.5

0.2 mM

Dithioerythritol (DTE)

NC

Calcium chloride in TBS, pH 7.2

10 mM

Dithiothreitol (DTT)

NC

Na-Carbonate/Na-Bicarbonate (0.2 M), pH 9.4

undiluted

Glucose

1 mM

Cesium bicarbonate

100 mM

2-Mercaptoethanol

1 mM

CHES, pH 9.0

100 mM

Thimerosal

NC

Na-Citrate (0.6 M),Na-Carbonate (0.1 M), pH 9.0

1:600 dilution*

Misc. Reagents & Solvents

Na-Citrate (0.6 M), MOPS (0.1 M), pH 7.5

1:600 dilution*

Acetone

1.0 %

Cobalt chloride in TBS, pH 7.2

NC

Acetonitrile

1.0 %

EPPS, pH 8.0

100 mM

Aprotinin

1 mg/L

Ferric chloride in TBS, pH 7.2

0.5 mM

DMF, DMSO

1.0 %

Glycine HCl (pH 2.8)

100 mM

Ethanol

1.0 %

Guanidine HCl

4M

Glycerol (fresh)

1.0 %

HEPES, pH 7.5

100 mM

Hydrazide

NC

Imidazole, pH 7.0

12.5 mM

Hydrides (Na2BH4 or NaCNBH3)

NC

MES, pH 6.1

100 mM

Hydrochloric acid

10 mM

MES (0.1 M), NaCl (0.9 %), pH 4.7

1:4 dilution*

Leupeptin

10 mg/L

MOPS, pH 7.2

100 mM

Methanol

1.0 %

Modified Dulbeccos PBS, pH 7.4

undiluted

Phenol Red

NC

Nickel chloride in TBS, pH 7.2

0.2 mM

PMSF

1 mM

PBS; Phosphate (0.1 M), NaCl (0.15 M) pH 7.2

undiluted

Sodium hydroxide

50 mM

PIPES, pH 6.8

100 mM

Sucrose

4%

RIPA lysis buffer; 50 mM Tris, 150 mM

1:10 dilution*

TLCK

0.1 mg/L

NaCl, 0.5 % DOC, 1 % NP-40, 0.1 %

TPCK

0.1 mg/L

SDS, pH 8.0

Urea

3M
1 mM

Sodium acetate, pH 4.8

200 mM

o-Vanadate (sodium salt), in PBS, pH 7.2

Sodium azide

0.2 %

Detergents

Sodium bicarbonate

100 mM

Brij 35

Sodium chloride

1M

Brij 56, Brij 58

1.0 %

Sodium citrate, pH 4.8 (or pH 6.4)

5
mM
mM)

CHAPS (CHAPSO)

1.0 % (5.0 %)

Sodium phosphate

100 mM

Deoxycholic acid

5.0 %

Tricine, pH 8.0

2.5 mM

Nonidet P-40 (NP-40)

5.0 %

Triethanolamine, pH 7.8

0.5 mM

Octyl -D-glucoside

0.1 %

Tris

50 mM

Octyl -D-thioglucopyranoside

5.0 %

TBS; Tris (25 mM), NaCl (0.15 M), pH 7.6

1:10 dilution*

SDS

5.0 %

Tris (25 mM), Glycine (192 mM), pH 8.0

1:10 dilution*

Span 2

1.0 %

Tris (25 mM), Glycine (192 mM), SDS (0.1 %), pH 8.3 undiluted

Triton X-100

5.0 %

Zinc chloride in TBS, pH 7.2

Triton X-114

(16.7

0.5 mM

Chelating agents

5.0 %

0.05 %

Triton X-305, Triton X-405

1.0 %

EDTA

0.5 mM

Tween 20, Tween 80

5.0 %

EGTA

NC

Tween 60

0.5 %

Sodium citrate, pH 4.8 (or pH 6.4)

5
mM
mM)

Zwittergent 3-14

NC

*Diluted with ultrapure water

NC: not compatible

(16.7

Methods for eliminating/reducing the effect of interfering substances

Remove the interfering substance by dialysis or gel filtration.
Dilute sample. This works only if the starting protein is sufficiently concentrated to remain in the working
range upon dilution.
Precipitate the proteins in the sample with acetone or trichloroacetic acid. The protein pellet is then
solubilized in ultrapure water or directly in the Working Reagent.
Increase the amount of copper in the Working Reagent (e.g. use a greater proportion of Reagent C; e.g.
Reagent A:B:C in a ratio of 25:24:2 or 25:24:3).
Note: For greatest accuracy, the protein standards must be treated identically to the sample(s).

Troubleshooting
Observation

Possible Cause

Precautions/Remedy

No color development

Chelating agents are present in Dialyze or desalt the sample.

the sample buffer
Dilute the sample.

Sample color less intense pH is altered by strong acid or Dialyze or desalt the sample.
than expected
alkaline buffer
Dilute the sample.
Sample color is darker than Protein concentration is too high
Dilute the sample
expected
Lipids or lipoproteins are present Add 2 % SDS to the sample to eliminate
in the sample buffer
interference from lipids
All the
purple

tubes

are

dark Reducing agents are present in Dialyze or dilute the sample

the sample buffer
Thiols are present in the sample Dialyze or dilute the sample
buffer

Persistent
precipitation Temperature of reagents is too Gently warm (~37 C)
all reagents
appears
after
Reagent low.
immediately prior to mixing. Or slightly
C(CuSO4)
is
added
to
dilute the Working Reagent with deionized
Reagents A+B
water
until
precipitate
disappears.
Also, the WR solution could simply be
filtered (the loss of reagents would be
negligible).

The Bicinchoninic acid Protein Quantitation

and Determination Products
Kit for Protein Quantitation and Determination

Bicinchoninic acid Protein Assay and Bicinchoninic acid Protein Assay Micro kits are formulations
based on bicinchoninic acid for the rapid and sensitive detection and quantitation of total protein content. The
Bicinchoninic acid method is faster and easier than Lowry, with much greater tolerance to interference from
non-ionic detergents and buffer salts.
The Bicinchoninic acid method combines the biuret reaction, i.e., the reduction of Cu2+ ions to Cu+ by proteins
in an alkaline medium with complexation of the latter with bicinchoninic acid. The purple-colored
Cu(bicinchoninic acid)23- complex displays a strong absorbance at 562 nm which is proportional to protein
concentration over a broad working range (20-2,000 g/ml for Bicinchoninic acid Protein Assay and 0.5-20
g/ml for Bicinchoninic acid Protein Assay Micro).
O

Protein + Cu2+

OH-

O
N

Cu+ + bicinchonic acid

N
+

Cu

(biuret reaction)
N
O

Purple Complex of Cu+ with BCA

Protein concentrations are generally determined with reference to standards of a common protein such as
Bovine Serum Albumin (BSA). If a more accurate quantitation of an unknown protein is required, the
calibration curve is constructed using a protein similar to the unknown.

Special features of the Bicinchoninic acid Protein Assay and Bicinchoninic

acid Protein Assay Micro

Photometric method (reading at 562 nm)

More sensitive than Lowry and less subject to interferences. In particular, it is insensitive to
detergents such as Triton-X 100 and SDS (1 %)

Linear working range for BSA is 20 - 2,000 g/ml for the Bicinchoninic acid Protein Assay and 0.5 - 20
g/mL for the Bicinchoninic acid Protein Assay Micro

Less protein to protein variation than Coomassie-based assays

Reagents are stable two years at room temperature

Adaptable to microplate format

References
1.) Smith, P.K. et al. (1985) Anal. Biochem. 150, 76-85 Measurement of protein using bicinchonininc acid.
2.) Walker, J. M. (1994) Methods. Mol. Biol. 32, 5-8.
3.) Pingoud, A, Urbanke, C., Hoggett J., Jeltsch, A., Biochemical Methods, pp.157-159, Wiley-VCH 2002.
Version WM100311