Protein estimation by Folin Lowry's method

Hello everyone,

Today I will be talking about  the most important type of chemical estimation for Proteins ie Folin lowry. The Lowry assay  is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on. The method is sensitive down to about 10 µg/ml and is probably the most widely used protein assay despite its being only a relative method , subject to interference from Tris buffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts. The incubation time is very critical for a reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is essential.

The determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. 

Requirements:

• 2% Na2C03 N NaOH
• 0.5% CuSO4 solution in 0.1% Na- K tartarate solution(Alkaline CuSO4)
• Folin -ciocaltaeau reagent
• Std protein (BSA)
• Distilled waater
• Unknown protein solution

The Folin–Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin–Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate . It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis.

The basic principle behind the experiment is very simple

The Nitrogen group present in protein reacts with copper ions under alkaline conditions and subsequent reduction of FC reagent to blue colour compound (heteropolymolybdenum blue)

Assay

1. Prepare a series  dilutions of BSA solutions are prepared by mixing stock BSA solution and water in the test tube  
2. Add 5.5 ml of Alkaline CuSO4 solution reagent in each tube.Mix the solutions well.
3. Incubate the tubes  for 10 min at RT, then cool to room temperature.
4. Then add 0.5 ml of reagent Folin Ciocalteau solution  to each tube and incubate for 30 min. 
5. Zero the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm.
6. Measure absorbance at 650 nm .
7. Plot the absorbance against protein concentration to get a standard calibration curve.
8. Check the absorbance of unknown sample and determine the concentration of the unknown sample using the standard curve plot.

-Dixy