Determination of Proteins Using Biuret and Lowry Assay Techniques Essay

Determination of Proteins Using Biuret and Lowry Assay Techniques - Essay Example Protein 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 mostly used due to its simplicity and less susceptibility 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 deep 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 pro tein estimation procedure even though highly susceptible to discerning compounds that interfere and distort solubility of insoluble proteins. The assay starts with copper ion complex that has peptide bonds, which are stabilized by tartrate in alkaline environment popular known as biuret chromophore. Gornall, Bardawill and David (1949) pointed out that biuret reaction is reduced 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 reduced blue 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 many interfering colors (Layne, 1957). In these two experiments, the basic law 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 following 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. Test tubes 6. Label 7. Test tube rack 8. Pipettes 9. Pipette bulb 10. Vortex mixer 11. Spectrophotometer 12. Cuvettes 13. Gelatin : 100Â µg cm-3 14. Globulin: 100Â µg cm-3 15. albumin: 200Â µg 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) and place them in a test tube rack. 3. Add water as provided in the instructions. 4. Prepare diluted Folin-Ciocalte u reagent and the Assay Mix. 5. Add 0.5cm3 of the protein solution to tubes (2 to 10). 6. Add gelatin 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.