Determination of Proteins Using Biuret and Lowry Assay Techniques Essay

Determination of Proteins using Biuret and Lowry Assay Techniques - Essay ExampleProtein assay is critical in the analysis of agricultural, industrial and biotechnological products. As argued by Bama et al. (2010), it is also important for research especially in analysis of enzymes, lectins and antibodies. This paper covers two kinds of assays used in quantitating total proteins. These includes the biuret and lowry techniques. Biuret assay, which is the least sensitive assay is among the coulometric methods (Quereshi et al. 2010). It is well-nighly used due to its simplicity and less power to chemical interference. The assay is dependent on polypeptide chelation of cupric iron in strong alkali. According to Mizuta et al. (2005), most biuret assays are used in samples containing 1 to 10mg protein/ml, which is then diluted five-fold by other reagents to form bass purple color. On the other hand, the Lowry method is a colorimetric assay that is based on folin-ciocalteau reagent and cupric ions of phenolic groups (Muyonga, Cole & Duodu, 2004). It is a popular protein estimation procedure even though highly open to discerning compounds that interfere and distort solubility of insoluble proteins. The assay starts with copper ion complex that has peptide bonds, which are stabilize by tartrate in alkaline environment popular known as biuret chromophore. Gornall, Bardawill and David (1949) pointed out that biuret reaction is rock-bottom under alkaline conditions of folin-ciocalteu reagent. Copper ions are used to enhance the reduction process. However, the principle chromogenic groups consist of the peptide linkages that lessen forbidding molybdotungstates, which catalyses polar amino acids, tyrosine and tryptophan. Nonetheless, the sensitivity of this test is based on protein composition and products of chemicals reaction resulting to the heteropolymolybdenum blue solution after being in absorbance condition of approximately 750nm, a wavelength that is out of range of some(prenominal) interfering colors (Layne, 1957). In these two experiments, the basic jurisprudence of light absorption, popularly knows as Beer-Lambert law is used to explain the linear relationship between protein (collagen) concentration and absorbance (Cliche, Amiot & Avezard, 2003). The yield of collagen is calculated using the side by side(p) lines equation Y=(VxC)/ W Where Yis the yield of collagen in mg/g Vis the volume of collagen solution in ml C is the concentration of the derived solution in mg/ml Wis the lyophilized weight in g Materials used 1. Protein sample of unknown concentration 2. Standard BSA 3. Distilled water 4. Lowry reagent 5. examination vacuum tubes 6. Label 7. Test tube rack 8. Pipettes 9. Pipette bulb 10. Vortex mixer 11. Spectrophotometer 12. Cuvettes 13. Gelatin cg cm-3 14. Globulin 100g cm-3 15. albumin 200g cm-3 Methods Lowry Technique Procedure 1. Prepare samples with up to 100 ?g of protein 2. Label the 9 test tubes as (1 to 10) an d place them in a test tube rack. 3. make for water as provided in the instructions. 4. Prepare diluted Folin-Ciocalteu reagent and the Assay Mix. 5. Add 0.5cm3 of the protein solution to tubes (2 to 10). 6. Add colloidal gel solution to tube 7 and 8 only. 7. Then add 2.5cm3 of solution D to each tube and mix well and leave the mixture at room temperature for approximately 10 minutes.