Protein Estimation

Dr. Kasturi Sarkar

Methods Used for Protein Estimation

Colorimetric assay
• Biuret Assay
• Folin-Ciocalteu (Lowry) Assay
• Bicinchoninic Acid (Smith) Assay
• Dye-Binding (Bradford) Assay

Ultraviolet Absorbance
Quantitative Determination of Proteins

• There is no completely satisfactory method to

determine the concentration of protein in any
given sample

• The choice of the method depends on the

 nature of the protein and the nature of the other
components present in the protein sample
 desired speed
 accuracy and
 sensitivity of assay
Primary Protein Structure
Secondary Protein Structure
-helix
-sheet
Tertiary Protein Structure
Quaternary Protein Structure
 Biuret Assay

Peptide Chains Biuret Complexes ( purple color )

Procedure

Sample conc. Sample vol (ml) Biuret reagent O.D at

(mg/ml) (ml) 545nm
BSA

1 1 4
2 1 4
4 1 4
6 1 4
8 1 4
10 1 4
Unknown 1 4
Blank Water (1) 4
Advantages

Reproducible
Very few interfering agents
(ammonium salts being one such agent )
Fewer deviations than with the Lowry or
ultraviolet absorption methods
Disadvantages

Requires large amounts of protein (1-20mg)

Low sensitivity
Reaction with Biuret
Folin-Ciocalteu (Lowry) Assay
Reagents
• Analytical reagent is prepared by mixing 2 ml of
(b) with 100 ml of (a)
(a) 50 ml of 2% sodium carbonate mixed with 50 ml of
0.1 N NaOH solution
(b) 10 ml of 0.5% copper sulphate, 5H2O and 1%
sodium potassium tartrate solution (stabilizes the
Cu2+ in alkaline solution).

• Folin - Ciocalteau reagent solution: this is a

solution of sodium tungstate and sodium molybdate
in phosphoric and hydrochloric acid.
Procedure
Sample Sample Alk. Lowry O.D. at
conc. vol (ml) CuSO4 reagent 660nm
(mg/ml) (ml) Incubatio (ml) Incubatio
BSA n at room n at room
temp for temp for
0.05 0.2 2 10 mins 0.2 30 mins
0.1 0.2 2 0.2
0.2 0.2 2 0.2
0.4 0.2 2 0.2
0.6 0.2 2 0.2
0.8 0.2 2 0.2
Unknown 0.2 2 0.2
Advantages

• Sensitive over a wide range, 100 times

more sensitive than Biuret assay and 10-
20 times more sensitive than UV detection

• Can be performed at room temperature

Disadvantages

• Many substances (Strong acids,

ammonium sulfate, reducing agents)
interfere with the assay

• Takes a considerable amount of time to

perform

• The assay is photosensitive, so illumination

during the assay must be kept consistent
for all samples
Bicinchoninic Acid ( BCA ) Assay
Procedure
Sample conc. Sample vol BCA reagent Incubation O.D at 562 nm
(mg/ml) (ml) (ml) at 60oC for
BSA 15 minutes

0.05 0.1 2
0.1 0.1 2
0.2 0.1 2
0.4 0.1 2
0.6 0.1 2

Prepare the required amount of protein determination reagent

by adding 1 volume copper sulfate solution to 50 volumes of
bicinchoninic acid solution.
Advantages

Very sensitive and rapid at elevated

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

The reaction does not go to completion

when performed at room temperature

Interferes with a no. of molecules sp with

reducing agents frequently used in protein
purification
Dye-Binding or Bradford Assay
Reagents

• Bradford Reagent (5x)

100 mg of Coomassie blue G250 in
dissolved in 50 ml of 95% ethanol. The
solution is then mixed with 100 ml of
85% phosphoric acid and made up to
200ml with distilled water. The reagent
should be filtered through Whatman
No. 1 filter paper and then stored in an
amber bottle at room temperature. It is
stable for several weeks.
Procedure

Sample conc. Sample volume Bradford reagent O.D. at 595nm

(mg/ml) (ml) (ml)
BSA

0.05 0.5 0.5

0.1 0.5 0.5
0.2 0.5 0.5
0.4 0.5 0.5
0.6 0.5 0.5
0.8 0.5 0.5
Unknown 0.5 0.5
Blank 0.5 0.5
Advantages

Fast, very sensitive and inexpensive

method
Highly specific for protein
Compatible with a wide range of
substances
Extinction co-efficient for the dye-protein
complex is stable
Non-linear standard curve over wide
ranges
Disadvantages

Response to different proteins can vary

widely, choice of standard is very important
CBBG primarily responds to arginine
residues (eight times as much as the other
residues). Hence for an arginine rich
protein, a standard should be found that is
arginine rich as well.
 Ultraviolet Absorbance

The absorbance of aromatic amino acids,

mainly tyrosine and tryptophan is measured at
280nm (O.D = cl)
and
the following equation is used to estimate the
protein concentration

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

 Procedure

Autozero spectrophotometer with the buffer

↓
Take the absorbance at 280 nm in a quartz
cuvette
↓
Change wavelength to 260 nm and zero with
water (or buffer)
↓
Take absorption at 260 nm in a quartz
cuvette
Advantages

• Very fast and inexpensive method

• Useful for estimation of protein before using

a more accurate method

• Sample can be recovered

Disadvantages

• It is the least sensitive of the methods

• Higher orders of protein structure, many other
cellular components, and particularly nucleic acids,
also may absorb UV light
• 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 influenced by pH and ionic
strength of the solution.
Points to be Remembered

• Use of clean glasswares and cuvettes

• Always warm up the spectrophotometer for 15-20

minutes before using

• Include a standard curve each time the assay is

performed

• Make sure your standard curve covers the

absorbance range of the unknown sample with at
least two points on either side
Summary
Biuret BCA Lowry Bradford Spectrophotometric
Assay Assay Assay Assay Assay
Accuracy Qualitative 0.5 μg/ml- 0.01- 2-120g/ml Detection of known
linear upto assay 1.5 mg/ml 1.0 mg/ml proteins only since molar
(1-20mg) extinction coefficients are
required

O.D at 540nm 562nm 660nm 595 nm 280nm

Interference Ammoniu Reducing Reducing Detergents, Chromophores that

m salts agents agents, basic absorb at 280nm
strong buffers,
acids, flavonoids
detergents,
Tris,
potassium
compds etc

Incubation 10 mins 30 mins 40 mins 5 mins nil

time