Estimation of protein by Folin Lowry method

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BACKGROUND

The Lowry method is one of the important methods of protein assay for determining the total amount of protein present in a given sample. So, the basic objective of this test is to estimate the amount of protein present in a sample by Lowry’s method.

The interaction between bio molecules is crucial for knowing their function. But the presence of the protein impurities can highly affect the result. Therefore, it is important to know the total protein concentration that includes both active and inactive forms of protein.

Principle:

The principle involved in Lowry method is determining the protein concentration by calculating the reactivity of the peptide nitrogen with the Copper ions under alkaline conditions followed by reduction reaction of Folinciocalteay phosphomolybdic phophotungstic acid to Heteropolymolybdenum blue by copper catalyzed oxidation of aromatic acids.

However, the method is highly sensitive to pH of the assay solution for which it should be strictly maintained at 10-10.5. But if very small volume of sample is used then pH will have least effect.

Buffers, sugars, nucleic acids, zwitter, lipids, and Sulphydryl reagents can also affect the Lowry procedure, so it’s essential to ensure that these substances are removed before starting the experiment.

The determination of protein concentration by Lowry method lab report, protein estimation by Lowry method pdf, protein estimation by Lowry method calculation, and protein estimation by Lowry method discussion are all important resources for understanding the results of this test and how to interpret them.¹

REQUIREMENTS

Sample: Assay sample

Reagents: Solution A: 2% sodium carbonate in 0.1 N NaOH

Solution B: 0.5% copper sulphate solution in 1% sodium potassium tartarate solution.

Lowry regent or alkaline copper sulphate solution: 50 ml solution A + 1 ml of solution B. Prepare it just before using.

Folin- ciocateau reagent- It is available commercially and is to be diluted just before using.

Standard protein solution: 200 mg of BSA is dissolved in 100 ml of distilled water in a volumetric flask.

Working standard: Dilute 10 ml of stock standard solution in 100 ml of distilled water.

Others: Volumetric flask, Test tubes

PROCEDURE

Take 0.2 ml of BSA working standard in 5 different test tubes and make the total volume up to 1 ml by using distilled water. Take another test tube and fill it with 1ml distilled water. It serves as blank. Now to the test tubes add 4.5 ml of reagent 1 and then incubate it for about 10 min.

After incubation add 0.5 ml of reagent II and then again keep it for incubation for about 30 min.

Now measure absorbance at 660 nm and make a standard graph. Measure the amount of protein present in the given sample from the drawn graph.

For drawing graph, plot the values of protein concentration in the X-axis and the absorbance value in the Y-axis. Now draw straight line through the points that will represent the value of absorbance drawn on the paper. From the absorbance value of the unknown protein draw perpendicular on the X-axis and measure the protein concentration of the given sample.¹

CONCLUSION

This is one of the common and popular method of estimating the total protein concentration in a given sample.

REFERENCES

Lowry, OH; Rosebrough, NJ; Farr, AL; Randall, RJ (1951). “Protein measurement with the Folin phenol reagent”. Journal of Biological Chemistry. 193 (1): 265–75.

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FAQs

What is the principle of estimation of protein by Folin Lowry method?

The principle behind the Lowry method is to determine protein concentration by calculating the reactivity of peptide nitrogen with Copper ions under alkaline conditions, followed by the reduction reaction of Folinciocalteay phosphomolybdic phosphotungstic acid to Heteropolymolybdenum blue through copper-catalyzed oxidation of aromatic acids. It’s important to note that the Lowry method is highly sensitive to the pH of the assay solution, which should be maintained at 10-10.5. However, if a very small volume of sample is used, the pH will have little effect.

How do you calculate protein in the Lowry method?

Measure absorbance at 660 nm and make a standard graph. And measure the amount of protein present in the given sample from the drawn graph.
For drawing graph, plot the values of protein concentration in the X-axis and the absorbance value in the Y-axis. Now draw straight line through the points that will represent the value of absorbance drawn on the paper. From the absorbance value of the unknown protein draw perpendicular on the X-axis and measure the protein concentration of the given sample.

Which reagent is used for protein estimation?

The Folin-Ciocalteu reagent is commonly used reagent for protein estimation.