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Lowry Method for Protein Estimation

The document outlines a laboratory procedure for determining protein concentration using the Lowry method, which relies on the reaction of peptide nitrogens with copper ions and the reduction of Folin-Ciocalteu reagent. It includes detailed steps for sample preparation, reagent preparation, and the calculation of protein concentration from absorbance readings. The procedure emphasizes the importance of maintaining specific pH levels and preparing reagents fresh before use.

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340 views2 pages

Lowry Method for Protein Estimation

The document outlines a laboratory procedure for determining protein concentration using the Lowry method, which relies on the reaction of peptide nitrogens with copper ions and the reduction of Folin-Ciocalteu reagent. It includes detailed steps for sample preparation, reagent preparation, and the calculation of protein concentration from absorbance readings. The procedure emphasizes the importance of maintaining specific pH levels and preparing reagents fresh before use.

Uploaded by

sonu bhai
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BT 312 Analytical Biotechnology Lab

Aim: Determination of Protein concentration by Lowry Method

Theory/Principle: The principle behind the Lowry method of determining protein


concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under
alkaline conditions and the subsequent reduction of the Folin-Ciocalteu phosphomolybdic
phosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of
aromatic acids tyrosine and tryptophan present in proteins. The intensity of the colour depends
on the amount of the aromatic amino acids present and will thus vary for different proteins.
The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be
maintained at 10 - 10.5.

Procedure:

1. Dilute the sample at a ratio of 1:25 (20uL sample in 480uL of milliQ).


NOTE: Dilution is an essential step at this particular ratio.
2. To the diluted sample (500uL), add 700uL of Lowry’s reagent (Preparation
mentioned below) in dark.
3. Incubate for 20 minutes in dark.
4. After incubation, add 100uL of Folin and ciocalteus reagent (Preparation mentioned
below) in dark.
5. Incubate for 30 mins in dark.
6. After incubation (aliquot 200uL in 96 well plate, quadruplicates), take reading at
750nm.

Reagents Preparation
Lowry’s Reagent

Lowry Reagent A
2% sodium carbonate in 0.1N sodium hydroxide

Dissolve 0.4gm sodium hydroxide and 2gm of sodium carbonate in 100mL of milliQ.

Lowry Reagent B
1% Sodium Potassium Tartarate in milliQ

Dissolve 0.1gm Sodium Potassium Tartarate in 10mL of milliQ.

Lowry Reagent C
0.5% of copper sulphate in milliQ.

Dissolve 0.05gm of copper sulphate in 10mL of milliQ

Lowry’s Reagent – Lowry reagent A + Lowry reagent B + Lowry reagent C in [Link] ratio.
Folin and ciocalteu reagent
45% Folin-ciocalteu reagent and 55% milliQ.

NOTE: Prepare Lowry’s reagent and Folin-ciocalteu reagent fresh before use.

BSA stock solution: 1mg/ml

Tabulation:

Sl. Conc. Of Volume milliQ Lowry’s Folin A750


No. BSA of BSA (ul) Reagent Ciocalteu (nm)
(ul) (ul) Reagent (ul)
A 1 mg/ml
B 500 ug/ml 250 (A) 250 700 100
C 250 ug/ml 250 (B) 250 700 100
D 125 ug/ml 250 (C) 250 700 100
E 62.5 ug/ml 250 (D) 250 700 100
Unknown - - 700 100
Sample

Calculation:

Prepare a standard curve of absorbance versus micrograms protein and determine the slope
y/x from the standard curve, which gives the A750 per unit of protein (μg). Hence determine
the amount of protein in the unknown sample.

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