Protein Determination Assays

Xin Li
Scott Group
05/10/2005

Quantitative Determination of Proteins

There is no completely satisfactory single
method to determine the concentration of protein
in any given sample

The choice of the method depends on the nature

of the protein, the nature of the other
components in the protein sample, desired
speed ,accuracy and sensitivity of assay

Methods Used for Protein Determination

Biuret Test
Folin-Ciocalteu ( Lowry ) Assay
Bicinchoninic Acid ( BCA ) Assay
Dye-Binding ( Bradford ) Assay
Ultraviolet Absorbance

Biuret Test

Peptide Chains

Biuret Complexes ( purple color )

Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949.
Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957
Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940.
Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269.
Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.

Biuret Test
Reproduciple
Very few interfering agents
(ammonium salts being one such agent )
Fewer deviations than with the Lowry or
ultraviolet absorption methods

Requires large amounts protein (1-20mg)

Low sensitivity

Biuret Test
1.

Warm up the spectrophotometer 15 min. before use.

2.

Dilute samples to an estimated 1 to 10 mg/ml with buffer. Add 1 ml to each

assay tube. Duplicate samples are recommended, and a range of dilutions
should be used if the actual concentration cannot be estimated.

3.

Prepare a reference tube with 1 ml buffer.

4.

Prepare standards from 10 mg/ml bovine serum albumin, preferably calibrated

using absorbance at 280 nm and the extinction coefficient. Range should be
from 1 to 10 mg protein.

5.

Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.

6.

Read at 550 nm.

Folin-Ciocalteu ( Lowry ) Assay

Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265. 1951.
Oostra, GM, NS Mathewson, and GN Catravas. Anal. Biochem. 89: 31. 1978.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
Hartree, EF. Anal Biochem 48: 422-427 (1972).

Folin-Ciocalteu ( Lowry ) Assay

Sensitive over a wide range

Can be performed at room temperature
10-20 times more sensitive than UV detection
Can be performed in a microplate format

Many substances interfere with the assay

(Strong acids, ammonium sulfate )
Takes a considerable amount of time to perform
The assay is photosensitive, so illumination during the assay
must be kept consistent for all samples
Amount of color varies with different proteins

Folin-Ciocalteu ( Lowry ) Assay

1. Add samples containing up to 100 g of protein.
2. Bring all tubes to 1 mL total volume with water.
3. Prepare the Assay Mix and diluted Folin-Ciocalteu reagent.
4. To each tube add 5 mL of assay mix and thoroughly vortex.
5. Incubate tubes at room temperature for 10 min.
6. Add 0.5 mL of diluted Folin-Ciocalteu reagent. Vortex immediately.
7. Incubate at room temperature for 30 min.
8. Vortex the tubes, zero the spectrophotometer with the blank and measure absorbance
at 500-750 nm.

Bicinchoninic Acid ( BCA ) Assay

P.K. Smith et al. (1985) Anal. Biochem. 150: 76.

K. J. Wiechelman et al. (1988) Anal. Biochem. 175: 231

Bicinchoninic Acid ( BCA ) Assay

Very sensitive and rapid if you use elevated temperatures

Compatible with many detergents
Working reagent is stable
Very little variation in response between different proteins
Broad linear working range

The reaction does not go to completion when performed

at room temperature

Bicinchoninic Acid ( BCA ) Assay

1. Prepare the required amount of protein determination reagent by
adding 1 volume copper sulfate solution to 50 volumes of
bicinchoninic acid solution.
2. Set up test tubes containing samples and known amounts of bovine
serum albumin in the range of 0 to 100 micrograms. Each tube
should contain 0.1 mL total volume.
3. Add 2.0 mL of the protein determination reagent to each tube and
vortex.
4. Incubate the tubes at 60oC for 15 min.
5. Cool the tubes to room temperature and determine the absorbance
at 562 nm.

Dye-Binding ( Bradford ) Assay

CBBG primarily responds to arginine residues

(eight times as much as the other listed resid
If you have an arginine rich protein,
You may need to find a standard
that is arginine rich as well.
CBBG binds to these residues in the anionic fo
Absorbance maximum at 595 nm (blue)
The free dye in solution is in the cationic form
Absorbance maximum at 470 nm (red).

Bradford, MM. A rapid and sensitive for the quantitation of microgram

quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

Dye-Binding ( Bradford ) Assay

Fast and inexpensive

Highly specific for protein
Very sensitive [1-20 g (micro assay) 20-200 g (macro assay)]
Compatible with a wide range of substances
Extinction co-efficient for the dye-protein complex is stable
over 10 orders of magnitude (assessed in albumin)
Dye reagent is complex is stable for approximately one hour
Non-linear standard curve over wide ranges
Response to different proteins can vary widely, choice of
standard is very important

Dye-Binding ( Bradford ) Assay

Absorption spectra of anionic and cationic forms of the dye overlap.

So the standard curve is non-linear although all kit providers of the
Bradford assay insist that the assay performs linearly.
The assay performs linearly over short concentration stretches.
If your sample is more than 20 micrograms, a second order curve
will fit much better than a linear curve.

Dye-Binding ( Bradford ) Assay

1. Warm up the spectrophotometer for 15 min. before use
2. Dilute samples with buffer to an estimated concentration of 1 to 20 micrograms/milliliter
3. Prepare standards containing a range of 1 to 20 micrograms protein (albumin or
gamma globulin are recommended) to a volume of 200 l (to a volume of 100 l if you
are adding 1 M NaOH)
4. Prepare unknowns to estimated amounts of 1 to 20 micrograms protein per tube to 200
l (100 l if you are using 1 M NaOH)
5. Add 100 l 1 M NaOH to each sample and vortex.
6. Add 800 l dye reagent and incubate 5 min.
7. Measure the absorbance at 595 nm.

Ultraviolet Absorbance
If you don't know what the protein concentration of an
unknown sample is likely to be, the ultraviolet method might
be a good starting point.
This is often used to estimate protein concentration prior to
a more sensitive method
Monitors the absorbance of aromatic amino acids, tyrosine
and tryptophan
Higher orders of protein structure, many other cellular
components, and particularly nucleic acids, also may absorb
UV light
This method is the least sensitive of the methods
The real advantages of this method are that the sample is
not destroyed and that it is very rapid.

Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 3: 447-455. 1957.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990.

Ultraviolet Absorbance
Quick
Sample can be recovered
Useful for estimation of protein before using a more accurate
method

Highly susceptible to contamination by buffers, biological

materials and salts
Protein amino acid composition is extremely important, thus the
choice of a standard is very difficult, especially for purified
proteins
Absorbance is heavily influence by pH and ionic strength of the
solution.

Ultraviolet Absorbance
Estimation Procedure
1.

Zero spectrophotometer to water (or buffer)

2.

Take the absorbance at 280 nm in a quartz cuvette

3.

Change wavelength to 260 nm and zero with water (or buffer)

4.

Take absorption at 260 nm in a quartz cuvette

5.

Use the following equation to estimate the protein concentration

[Protein] (mg/mL) = 1.55*A280 0.76*A260

BioRad DC Protein Assay

Based on Lowry
Assay with following
improvements:

1. Reaches 90% of its

maximum color
development within 15
minutes
2. The color changes not
more than 10% in 2
hours

BioRad DC Protein Assay

Prepare 5 dilutions of samples and 5 dilutions of a protein

standard containing from 0.2 mg/ml to about 1.5 mg/ml protein. A
standard curve should be prepared each time the assay is
performed. For best results, the standard should be prepared in
the same buffer as the sample.
BSA
mg/ml

0.24
(1/6)

Pf cell 0
extract

0.48
(2/6)

0.72
(3/6)

0.96
(4/6)

1.20
(5/6)

1.44
(6/6)

0.1C

0.2C

0.3C

0.5C

BioRad DC Protein Assay

Pipet 5 ul of standards and samples into a microtiter plate

Add 25ul of reagent A into each well
Add 200ul reagent B into each well
Agitate the plate to mix the reagents
1

0.24

0.48

0.96

1.20

1.44

empty

0.24

0.48

0.96

1.20

1.44

empty

0.24

0.48

0.96

1.20

1.44

empty

blank

blank

blank

blank

blank

blank

empty

0.1C

0.2C

0.3C

0.5C

empty

0.1C

0.2C

0.3C

0.5C

empty

0.1C

0.2C

0.3C

0.5C

empty

empty

empty

empty

empty

empty

empty

empty

Empty= Air
Blank=Reagent A+B
0=Reagent A+B+5ul water

BioRad DC Protein Assay

After 15 minutes, absorbance can be read at 750nm.
The absorbance will be stable for about 1 hour

From Oliver

BioRad DC Protein Assay

1

0.037

0.049

0.060

0.059

0.087

0.042

0.051

0.070

0.069

0.088

0.039

0.051

0.071

0.085

0.087

0.028

0.050

0.053

0.124

0.161

0.029

0.047

0.076

0.097

0.173

0.018

0.049

0.067

0.104

0.158

From Microplate reader in Bioexpress Lab

BioRad DC Protein Assay

Standard

0.037

0.049

0.060

0.059

0.087

0.042

0.051

0.070

0.069

0.088

0.039

0.051

0.071

0.085

0.087

Average

0.0393

0.0503

0.0670

0.0710

0.0873

Con.

0.24

0.48

0.72

0.96

1.44

Plot Concentration as Y axis

Plot Concentration as X axis

Standard

0.1

1.5

0.08

VS

1
0.5

y = 19.815x - 0.2861
R2 = 0.9868

0
0

0.02

0.04
0.06
Absorbancd

0.08

0.1

y = 19.815x - 0.2861
[Protein]= 19.815[A]- 0.2861

Absorbance

Concentration

Standard

0.06
0.04
y = 0.0498x + 0.015
R2 = 0.9868

0.02
0
0

0.5

1
Concentration

1.5

y = 0.0498x + 0.015
[Protein]=([A] -0.0015)/0.0498

Could introduce errors into the calculation

BioRad DC Protein Assay

[Protein]= 19.815[A]- 0.2861
1

0.028

0.050

0.053

0.124

0.161

0.029

0.047

0.076

0.097

0.173

0.018

0.049

0.067

0.104

0.158

Average

0.0250

0.0487

0.0653

0.1083

0.1640

Con.

0.21

0.68

1.01

1.86

2.98

Fraction

0.1 protein 0.2

0.3
concentration

0.5

concentration

Standard

3.5
3
2.5
2
1.5
1
0.5
0
-0.5 0

y = 3.2305x - 0.0644
R2 = 0.9741
0.2

0.4

0.6

0.8

1.2

fraction

Protein Concentration= 3.1661mg/ml

Tips
Use clean glassware and supplies
Make sure cuvettes are clean of all residues
Protein assays are strongly influenced by the
composition of the proteins present in your sample
Become familiar with spectrophotometry before
proceeding
Always let a spectrophotometer warm up for 15-20
minutes before using
Know the limits of the spectrophotometer with which you
are using

Tips
Standard curves are not always linear
The protein used for your standard curve must make
sense
Make sure your standard curve covers the absorbance
range of your unknown with at least two points on either
side
Make sure that your protein solution behaves in a
reproducible manner to the assay method by making a
dilution curve
Use buffer and water blanks to anchor down your standard
curve
Place the protein concentration on the y-axis of you
standard curve plot so that you can use the best-fit
equation directly for concentration determination