Methods for protein determination

1. Lowry (Folin) protein assay

2. Spectrophotometry based on UV
absorption

3. Bradford protein assay

4. Biuret test
Bradford method
use of coomassie G250 dye in a colorimetric
reagent for the detection and quantitation of
total protein.
In the acidic envirnment of the reagent
protein binds to the coomassie dye
This results in aspecial shift from the
reddish/brown form of the dye absorbance
maximum at 465nm to the blue form of the
dye absorbance maximunm at 610nm
 The differences between the two forms of the dye (bound and
unbound) is greatest at 595nm, so that is the optimal
wavelength to measure the blue color from the coomassie dye
protein complex.
development of color bradford protein assays has been
associated with the presence of certain basic amino acids
primarily arginine, lysine ,histidine in the protein.
Free amino acids, peptides and low molecular weight proteins
dont produce color with coomassie dye reagents, unbound
forms are green or red.
it is highly sensitive, is able to measure 1-20 g of protein and is
very fast.
 Procedure
Test Sample Sample vol., Water, Bradford reagent,
l l l

Blank 0 200 800

BSA Standard 20 180 800
(10 g/ml)

BSA Standard 40 160 800

(20 g/ml)

BSA Standard 60 140 800

(30 g/ml)

BSA Standard 80 120 800

(40 g/ml)

BSA Standard 100 100 800

(50 g/ml)

Protein Sample 100 100 800

Incubate the tubes for 5 mints at room temperture.

Read at 595 nm on spectrophotometer.
Make the standard curve and measure the concentration of unknown.

Blank solution: A blank solution is a solution containing no analyte of

interest, usually used to calibrate instruments such as a colorimeter.
How to calculate the concentration

According to beer_law The Beer-Lambert law (Beers law)

mathematically establishes the relationship between concentration
and absorbance in many photometric determinations. Beers law is
expressed as
A = abc
The concentration of substance is directly proportional to the amount of
light absorbed or inversely proportional to logarithm of the transmitted
light.
A: absorptivity constant for the substance
B: length of the light path through the substance.
Samples treated with the Bradford
assay. The brown sample (lower
absorbance) contains no protein,
while the blue sample (higher
absorbance) contains protein.
The amount of protein in the
second sample can be determined
by comparison to a standard curve