Sunday, July 26, 2009

what is it with grade

Quantitative determination of protein


In general, there is no completely satisfactory single method to determine the concentration of protein in any given sample. The choice of the method is depend on the nature of the protein, the nature of the other components in the protein sample and the sensitivity of assay. Several of the methods commonly used for protein determination are such as Biuret Test, Lowry Assay, Bicinchoninic Acid (BCA) Assay, Bradford Assay, Spectrophotometric Assay and also dry weight. But commonly used in the laboratory experiment are Biuret Test and Lowry Assay.

Materials and Methods

To determine the grade of samples of several type of egg, we need to plot a standard curve of absorbance versus concentration of protein in mg/L. In that experiment, Lowry Assay had been used. 0.25mL of protein had been mixed with 2.5mL of Lowry reagent 1. After 10 minutes, 0.25mL of Lowry reagent 2 had been added and mixed well immediately. After 30 minutes, the absorbance at 760nm had been measured. All this method were been applied to all 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/L of gelatin. The standard curve had been plotted. The absorbance of test sample had been measured.

Graph 1: Actual curve

Graph 2: Theoretical curve

Table 1: The absorbance of sample eggs


By study at the curve above, the actual curve was differed with the theoretical curve. Although the Lowry total protein assay has withstood the test of time, there are several features of the assay that have to be kept in mind. Because these methods rely on the presence of readily oxidizable amino acids such as tyrosine, cysteine, and tryptophan there is a variation in response from proteins with differing amino acid content. Therefore it is advisable that the protein used for generating the standard curve be consistent from experiment to experiment. Likewise, an overabundance of the amino acids in relation to the assay reagents, as would occur with high protein level, will result in a loss of linearity of the assay. In extreme cases this will lead to a precipitation of the chromogens and loss of color prematurely. Likewise, the assay color is only stable for approximately one hour, after which a similar phenomenon occurs in samples with normal concentrations.

When we run an assay we must ensure that only the substance we are assaying is responsible for absorbance of light in the wavelength range of interest. All conditions under which standards and unknowns are prepared should be kept identical. If solutes in the sample buffers affect absorbance, then we have a problem. We won't obtain accurate results if we vary the volumes in which we prepare and assay standards and unknowns. The timing of reading absorbance, temperature at which we keep the materials, and all other physical factors should be kept the same. Because it is not always practical to use identical buffers for all unknowns and standards, we need only ensure that none of the components of any of the buffers has a significant effect on absorbance.


Because of the curve that is not same as the theoretical, and we can conclude that the experiment is failed, so the grade of the sample of eggs cannot be determine as their absorbance cannot be compared.


1. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.

2. Robert L. Switzer and Liam F. Garrity (1999) Experimental Biochemistry. W.H Freeman and Company

To respond about the relationship between protein content and the grade of egg, yes, the quality is indicated by egg grade, not size. A process called candling, in which eggs are held in front of a light source, allows egg graders and inspectors to observe the size of the air pocket (an indicator of quality and age), the condition of the yolk and the white, and any hidden defects. These factors, along with shell soundness, cleanliness and shape, are used to classify eggs as Grade AA, Grade A or Grade B. AA and A are the grades most commonly marketed at retail. Grade AA eggs are the highest quality, but there is no difference in nutritive value among the different grades.