Tuesday, August 25, 2009

Biuret Assay

The Biuret reaction was one of the first for the determination of protein concentration. It remains as a rapid determination, but is not very accurate. It is useful during protein separation procedures since there are fewer salt interference reactions than with the Bradford or Lowry techniques. The color formed is stable for about 1-2 hours and consequently all spectrophotometer readings must be made as soon as possible after the incubation step.
The Biuret reaction involves a reagent containing copper (cupric) ions in alkaline
solution. Molecules containing 2 or more peptide bonds associate with the cupric ions to form a coordination complex that imparts a purple color to the solution with Amax = 540 nm. The purple color of the complex can be measured independently of the blue color of the reagent itself with a spectrophotometer or colorimeter.
Protein assays based on the Biuret reaction were developed and used by numerous
investigators throughout the first half of this Century. Typically each investigator adapted the basic reaction to suit their own needs, introducing their own variations of concentrations and assay conditions. Thus there are many versions of Biuret assay in the earlier literature.


0.039 1

0.089 2

0.096 3

0.119 4

0.154 5

0.236 6

Lowry Assay

The Lowry method depends on the presence of tyrosine within the protein to be measured. The standard protein must contain approximately the same number of tyrosine residues as the sample, or the procedure will be inaccurate. If there are no tyrosine residues in the sample to be measured, the Lowry method of protein determination is useless, and use should be made of the Bradford assay. In general, the Bradford assay is the method of choice for protein determinations.
A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. It can be used in many biological experiments
The assay is first performed with various known concentrations of a substance similar to that being measured. For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin. The assay procedure may measure absorbance, optical density, luminescence, fluorescence, radioactivity, or something else.

Experiment 2
( Application of protein : Making a protein glue )
Cows milk make a glue. The protein in the milk can be used to make glue. The protein in the milk is casein. So, in reality, cows make milk, but you can use milk to make glue. Casein is actually a micelle consisting of a protein subunit that somehow stabilizes the micelle so that it limits its growth and stays dispersed in the milk colloid. The other components of casein are calcium and phosphate ions. When the protein subunit is removed from the casein micelle, they can clot together to form the curd that can be further treated to make cheese or an adhesive suitable for use in paper, plastics, or glues. When mixed with lime, the curds form a material know as whitewash, which was used in colonial times to paint houses.

Elmer’s glue used to be made from casein, however, it is now made from a polymer, polyvinylacetate (PVA) since that polymer is more stable and has a long shelf storage life. Elmer’s blue gel glue is made from polyvinylalcohol.


3 alternative methods determine protein

Kjeldahl method
A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration.
Enhanced Dumas method
Recently, an automated instrumental technique has been developed which is capable of rapidly measuring the protein concentration of food samples. This technique is based on a method first described by a scientist called Dumas over a century and a half ago. It is beginning to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness.
Measurement of Bulk Physical Properties
Density: The density of a protein is greater than that of most other food components, and so there is an increase in density of a food as its protein content increases. Thus the protein content of foods can be determined by measuring their density.
Refractive index: The refractive index of an aqueous solution increases as the protein concentration increases and therefore RI measurements can be used to determine the protein content.

What is an “appropriate blank” and why?

Appropriate blank is known as zero spectrophotometer which is a good way in determining protein concentrations by measuring the absorbance at different rate. Appropriate blank exclude other substances from spectrophotometer reading without removing them the experimental tube. But, one of the problem is other substances will interfere the absorbance reading.