A test sample diluted with 85% Sodium Chloride Solution is used to reach a final concentration of 150-100 ug/ml. The blank test tube will be added with 0.20ml 85% Sodium chloride Solution. The blank test tube will be used as reference test tube. This will be the test tube for comparison purposes with the other test tubes containing the various reagents. The blank test tube will be the first tube to be read in the spectrophotometer that will set back the reading to zero. All the other test tubes would be read in comparison with the blank test tube’s reading to determine significant results and readings.
The rest of the test tubes would be added with certain volume of reagents such as Biuret reagent and Folin and Ciucalteu’s reagent, mixed and allowed to stand for different holding times. The contents of the test tubes would then be read with the absorbance with the blank solution as reference. The protein concentration of every diluted sample would be determined. The use of Biuret Method is greatly used for protein analysis. Biuret is a compound formed from combination of urea through heat producing amide groups.
A peptide bond results with the amide group of the biuret reagent and the carboxyl group. This would result to blue color solution observable through spectrophotometer in the presence of copper ion complex (Biuret Protein Assay, 2010). The assay would greatly result to colored solution in alkali environment (Detection and Assay Methods, 1995). One of the reagents used in the experiment is the Folin and Ciocalteu’s Phenol reagent. This mixture of phosphomolybdate and phosphototungstate (MP Biomedicals, 2014) is useful a reagent commonly used for portein concentration determination through the Lowry method.
The addition of this reagent to a previously pretreated diluted sample generates chromogens that increase absorbance to 550-750 nm. Color intensity is affected by the presence of tryphtophan and tyrosine (Sigma-Aldrich, 2014). Under alkaline conditions, a monovalent ion and radical groups of tryhtophan and tyrosine reacts with the Folin reagent turning the solution into tungsten blue that can be viewed on spectrophotometer (Caprette, 2012). Results and Discussion In accordance with the procedures for this protein analysis, the results are can be summed up with this table and graphical representation: Table 1: Protein Analysis Test Tube Absorbance Reading ug/ml Protein Concentration 1 0 0 2 0.28 250 3 0.53 500 4 0.74 750 5 0.96 1000 Graph 1: Protein Analysis Based on the results it can be observed that the absorbance and protein concentration is linear in nature.
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