Estimation of Protein by Lowery Method

9 EXPERIMENT NO: 5 DATE: 17.11.2022 ESTIMATION OF PROTEIN BY LOWRYMETHOL AIM TO determine the concentration of proteins by howry's method PRINCIPLE The howhy method is one of the common and popular method of estimating the total protein concern- tration in a given sample It uses copper which bonds with the pepticle bonds in proteins under alkali ne conditions. This forms a monovalent copper ion which can then reaut with the Folin ciocaltaeu reagent which in turn can be reduced into a blue colored substance This blue color can be measured using a spectrophotometer to determine the concentration of blue in the sample, at an absorbance of 660 nm. MATERIALS REQUIRED Test tube, micro pipette, test tube stand, standard BSA solution, alkaline copper solution, Folin ciocalt- alu reagent, two unknown solution, distilled water, Reagent A, Reagent B PROCEDURE 1. First seven test tubes were cleaned and marked as blank, S1, S2, S3, S4, S5 ,T, and T2 (unknown solution) 2. In theblank tube distilled water was only added, followed by alkaline copper sulfate solution and Folin -cioialteau reagent. 3. In the tubes marked as T1 14 T2, two unknown samples of protein which are of different concentration was added followed by 2ml of distilled water, 5 ml of ackaline CUSOA solution and 500M1 of Folin - ciocalteau reagent

10 A. were out from the tubes the volume S1,S2 0.2,0.4,0.6,0.8 and 1.0 ml of the protein marked solution as S3, SA pipetted and S5 and it was made up to 5. added, mixed well and allowed It to stand TO each tube 5 ml of alkaline (USO4 solution were at 3ml by adding distilled water room temperature for 10 minutes. 6. Then to each tube 0.5 ml of Folin ciocaltaen reage me was added and mixed with the help of vortex 7. After working, all the tubes were kept in a dark 8. Then measured the absorbance of the purple blue place for 20 minutes colour at 660nm. 9. Readings of each tube were noted and a graph was plotled 10. Protein concentration of the given unknoun sampl were calculated from the graph. RESULTS The concentration of protein in the given unknown sample (TD is 74 mg/ml The concentration of protein in the given unknown sample (T2) is 20 Mg/ml J. 24/11/22 r