Materials and Apparatuses for Biuret Test - Lab Report Example

Protein analysis The experiment was conducted to determine the level of protein in different sets of solution. In the conduction of the experiment, two methods of protein analysis were employed, i.e. the biuret protein analysis and the Folin protein assay methods. Working with the two methods, the results of the experiment indicated that biuret protein analysis methods is a more definitive method of protein determination as the concentration mean of protein using this method was 1.88 x 10-3 while Folin protein assay methods had a standard deviation of 0.026. Introduction Biuret test is a chemical test used in the determination of the presence of a peptide bond. In the course of the experiment, the presence of a peptide bond through the application of the method would result to a copper (ii) ion forming violet color in an alkaline solution. The Biuret test in this experiment is used in the determination of the concentration since peptide bonds occur with the same frequency in each amino acid in the peptide. This is possible in this experiment since the absorption at 550 nm is directly proportional to the protein concentration in accordance to Beer-Lambert law, thus enabling the experiment to determine the level of protein concentration in a solution (Janairo et al. 2011). Folin protein assay is a biochemical assay for determination of the total level of protein in a solution. In this experiment, the concentration of protein is manifested in the color change in the sample solution in proportion to the concentration of protein. The method is based on the reaction of Cu+ produced by the oxidation of the peptide bonds (Lowry et al. 1951). Materials and methods Materials and apparatuses for Biuret test 1. Protein samples of unknown concentration 2. Distilled water 3. Biuret reagent 4. Test tubes 5. Labels 6. Pipets 7. Test tube racks 8. Pipet bulb 9. Vortex mixture 10. Spectrophotometer 11. Cuvettes 12. Standard BSA (5g/l) Method 1. Label the test tubes as (1 to 19) and put them in a test tube rack 2. Add to pairs of test tubes i.e. (tube 1&2, 3&4, 5&6 etc.) the solution starting with Protein 3mg for 1&2 and reducing the amount by 0.5 for each subsequent pair till tubes 11&12 3. Tubes 13,14&15 are put in triplet with each unknown amount of solution thus called X while tubes 16,17&18 equally each hold unknown amount of solution and called Y 4. Thoroughly mix the solutions by vortex mixer and allow 30 minutes standing 5. Read the absorption of each tube against the blank at 550 nm 6. Calculate protein concentration for each tube of standard 7. Record the findings in a table 8. Calculate mean Materials and apparatuses for Folin protein assay test 1. Copper solution 2. Folin Ciocalteu reagent 3. Protein standard (2mg/ml BSA) 4. Distilled water 5. Test tubes 6. Labels 7. Test tube racks 8. Vortex mixture 9. Spectrophotometer 10. Cuvettes Method 1. Add distilled water to the protein standard to make 1mg/ml solution 2. Prepare a range of protein standard with the dilute BSA 3. Label two sets if the tubes with 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, unknown X, and unknown Y 4. Add 0.05 ml of the standard to the tubes 5. Add 0.1ml copper solution to the tubes and gently mix by Vortexing 6. Add 1ml Folin Ciocalteu reagent and incubate at room temperature for 15 minutes 7. Turn on the Spectrophotometer to warm up, adjusting the wavelength to 650-700nm 8. Add 1ml distilled water to Cuvettes to zero the absorbance of Spectrophotometer. Measure the absorbance of each tube and record the results Results For the biuret protein assay, when 3mg protein is used in the experiment, the average absorbance in 550nm is 0.690. For 2.5 mg, the average absorbance is 0,440nm, for 2mg protein, the average absorbance is 0.588nm. When 1.5 mg protein is used, the average absorbance is 0.564 nm, 1mg result to average absorbance of 0.540nm while 0.5ng solution results to an average absorbance of 0.224nm. In tube X the average absorbance is 0.454nm while tube Y which contains approximately half of the volume of X results to an average absorption of 0.232nm. From the experiment, it is evident that the amount of absorption is directly proportionate to the amount of protein concentration in a solution at a standard absorption level of 550nm absorbance. Thus, the biuret protein assay test is a concrete test that can be used in the determination of the peptide bonds and the amount of proteins in a solution (Lipscomb et al. 2006). Biuret protein assay test results For the Folin protein assay test, the results indicated that in a 0.12 mg protein, the average absorbance at 660nm was 0.763, at 0.1 mg protein, average absorbance is 0.583. In 0.08 mg proteins, the average absorbance at 660nm is 0.596, at 0.06 mg protein, the average absorbance is 0.460 while at 0.04 mg protein, and the average absorbance is at 0.325. At 0.02 mg protein, the average absorbance at 660nm is 0.195 and in tube X, the average absorbance is 0.481 while in tube Y, which has a slightly higher amount of solution than tube X, the average absorbance at 660nm, is 0.551. The result of this experiment indicates that when using Folin protein assay test, the color change in the sample solution in proportion to the concentration of protein. Folin Protein assay results Calculations Biuret protein assay test From The Graph X = 1.35 1- Concentration= 1.35 x 2.5= 3.37 MG/ML 2- Mean= = 0.454 3- Standard Deviation= = 8.73 x 10-3 4- Standard Error Of Mean= SEM= SEM= 5.04 x 10-3 From The Graph Y = 0.62 1- Concentration= 0.62 x 2.5= 1.55 2- Mean= = 0.232 3- Standard Deviation= = 1.88 x 10-3 4- Standard Error Of Mean= SEM= SEM= 1.05 x 10-3 Folin protein assay test From The Graph X = 0.066 5- Concentration= 0.066 x 2.5 x 20= 3.3 MG/ML 6- Mean= = 0.481 7- Standard Deviation= = 0.026 8- Standard Error Of Mean= SEM= SEM= 0.015 From The Graph Y = 0.075 5- Concentration= 0.075 x 2.5 x 20= 3.75 6- Mean= = 0.551 7- Standard Deviation= = 0.025 8- Standard Error Of Mean= SEM= SEM= 0.014 Discussion The biuret reagent is composed of sodium hydroxide (NaOH) and hydrated copper (II) sulfate as well as potassium sodium tartrate. The potassium sodium tartrate is added to stabilize the cupric ions. Cupric ions react with nitrogen atoms in the peptide bond leading to the displacement of the peptide hydrogen atoms under alkaline condition. This then results to differentiation of the color in the copper (II) sulfate solution in relation to the concentration of the proteins in the solution. In Folin protein assay test, the method utilizes the mechanism of reaction of copper ion with the peptide bond under alkaline condition, (biuret teas) with the application of oxidation of aromatic protein residue. The method is best applicable in a protein concentration of 0.01-1.0mg/ml and bases on the reaction of copper ion produced by peptide bond oxidation with Folin-Ciocalteu reagent (Everette et al. 2010). The concentration of the reduced Folin reagent is measured by absorbance at 660nm. The more the proteins and amino acids concentrated in a solution, the more Folin is absorbed. References Everette, J.D. et al., 2010. Thorough study of reactivity of various compound classes toward the folin-Ciocalteu reagent. Journal of Agricultural and Food Chemistry, 58, pp.8139–8144. Janairo, G. et al., 2011. Determination of the sensitivity range of biuret test for undergraduate biochemistry experiments. e-Journal of Science & Technology, 5, pp.77–83. Lipscomb, I.P. et al., 2006. The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. Journal of Hospital Infection, 64, pp.288–292. Lowry, O.H. et al., 1951. Protein measurement with the Folin phenol reagent. The Journal of biological chemistry, 193, pp.265–275. Read More