Lowry method

Practical No 2
Estimation of Protein by Lowry et al Method.

Introduction:
Proteins are essential macromolecules that perform a wide array of functions

in living organisms. Composed of long chains of amino acids, they
contribute to cellular structure, catalyze biochemical reactions as enzymes,
transport molecules, and play a key role in the immune system. The
sequence of amino acids in a protein determines its unique three-dimensional
structure, which in turn defines its specific biological function. Proteins are
crucial for numerous physiological processes, including cell signalling, gene
regulation, and tissue repair. Protein quantification is a vital step in
biochemical and molecular biology research, helping to determine protein
concentrations in various samples. Several methods like Biuret Method,
Lowry Method, Bradford Method, etc. are available for protein estimation.
Principle:
The Folin-Lowry method is one of the commonly used methods to determine

the concentration of proteins in a sample. This assay is about 10 times more
sensitive than the Biuret method. Its principle is similar to that of the Biuret
method, except that here a second reagent (ie., the Folin- Ciocalteau reagent)
is added to enhance the color produced, which depends on the amount of
protein present in the sample. The two sequential reactions accounting for
the intense blue color are: (a) the biuret reaction answered by the protein
when reacted with the alkaline cupric sulfate and (b) the reduction of the
Folin-Ciocalteau reagent (phosphomolybdate-phosphotungstate) by aromatic
amino acid residues (tryptophan, tyrosine, etc.) present in the protein. The –
CO-NH- bond (peptide) in polypeptide chain reacts with copper
sulphate in an alkaline medium to give a blue colored complex. In
addition, tyrosine and tryptophan residues of protein cause reduction of
the phosphomolybdate and phosphotungstate components of the Folin-
Ciocalteau reagent to give bluish products which contribute towards
enhancing the sensitivity of this method. The color intensity produced
depends on the aromatic amino acids present, and will thus vary for different
proteins. The developed blue-color absorbs maximally at 660 nm. Bovine
serum albumin (BSA) is used to prepare as the standard stock solution as it is
rich in aromatic amino acids which respond to this reaction. The Lowry
method is sensitive to low concentrations of protein.
Reagents:
A: 2% Na2CO3 in 0.1N NaOH
B: 0.5% CuSO4 in 1% Na-K-tartrate solution
C: Solutions A and B mixed in the ratio 50:1 just before use
D: Commercially available Folin- Ciocalteau reagent diluted with DW in the

ratio of 1:1 just before use.
2. Prepare a stock solution having 500 µg/ml of BSA in distilled water by

adding 50 mg BSA to-100 ml distilled water. Now, set up a series of BSA
solutions in the concentration range of 0-500 µg/ml by mixing distilled water
with the stock solution as indicated in the table.
Procedure:
1. Pipette out the standard protein solution in different volume (0.2-1 ml) in
a series of test tube.
2. Add distilled water in all the test tubes upto 1 ml.
3. Add 4 ml of solution C to all the tubes and incubate them for 10 min at
room temperature.
4. Add 0.5 ml of solution D (1:1 diluted Folin’s reagent) into each tube, mix
well and incubate in the dark for 30 min.
5. Using the blank, set zero absorbance at 660 nm and read the absorbance
for all the tubes. Record the results.
Observation Table:
(Conc. of std. Solution: 500 µg/ml)
Sr. Conc. Conc. D/ W Alk. Incubatn FCR Incubatn Abs.
No. of Std. of Std. CuSO4 Reagent 660nm
BSA BSA (ml)
(ml) (µg/ml)
1 0.0 1.0
2 0.2 0.8
3 0.4 0.6
4 0.6 0.4 5 min at
30
4 ml room
5 0.8 0.2 0.5 ml mins. in
temperatu
6 1.0 0.0 dark
re
S (I) 1.0 ? 1.0
S 1.0 ? 1.0
(II)
Calculation:
Concentration of unknown Protein solution:
O . D of sample
X Concentration of Standard
O. D . of Standard
Result:
1. By Calculation:
Concentration of unknown Protein solution is ___________µg/ml.
2. By Graph:
Concentration of unknown Protein solution is ___________µg/ml.

Lowry method

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Practical No 2

Estimation of Protein by Lowry et al Method.

Proteins are essential macromolecules that perform a wide array of functions

The Folin-Lowry method is one of the commonly used methods to determine

A: 2% Na2CO3 in 0.1N NaOH

B: 0.5% CuSO4 in 1% Na-K-tartrate solution

C: Solutions A and B mixed in the ratio 50:1 just before use

D: Commercially available Folin- Ciocalteau reagent diluted with DW in the

2. Prepare a stock solution having 500 µg/ml of BSA in distilled water by

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