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Saturday, November 21, 2009

Folin-Lowry Method Estimation of protein

Principle:-

Protein reacts with the Folin Go Chateau reagent to form a colored complex. The co lour is so formed due to the reaction of alkaline ca++ with protein as in the Burette method and the reduction of Phosphomolybdate
[ Mo+6-----> Mo+4 ] by tyrosine and tryptophan present in protein. The intensity of co lour depends upon the amount of these aromatic amino acids present and thus will vary for different proteins.

Requirements: - Standard protein solution (0.2 mg/ ml)
Alkaline caso4 solution, Folin – C io ch√Ęteau reagent.

Procedure: - Standard curve.
Prepare a series of test tubes containing varying amounts (0.1 to 0.5 mg) of protein. Making the volume to 3 ml with distilled coater. Add 3ml of alkaline cuao4 som. Mix well and allow to stand for 15 mins. Add 0.5ml of Folin ciocalteau, reagent, and mix well and allow standing for 30 mins.at room temperature. Measure the absorbance or Extinct calorimetrically against concentration.
Isolation of allow in from egg.
Crock an egg. Separate the yolk and add the egg white to 250 ml of distilled water in a beaker. Stir briskly when the globulins separate out as the ppt and albumins remain in solution. Make up the volume to 250 ml with distilled water. Centrifuge 10 ml of this solution at 2000 r.p.m. for 10 min. Dilute 25 ml of this solution to 100 ml and use 2 ml for co lour development. Determine the concentration of albumin in the sample. Express the conc. As gm. of albumin per egg.

Note:-
Folin-Lowry Method is more sensitive and is best applicable with in the range of 0.01 mg to 1.0 mg of protein concentration. Berate method is best applicable within the range of 1.0 mg to 10 mg of protein concentration.
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