University:
Swansea University
Course:
PM134 | Microbiology
Academic year:

2023
Views:

351

Pages:

2
Author:

Sanjeev K

EXPERIMENT NO:5 DATE: 17-11-2022 ESTIMATION OF PROTEIN BY LOWRY METHOD AIM To determine the concentration of proteins by hewnj's method PRINCIPLE The Lowry method is one of the common and Popular mearned of estimating the total protein concern- tration in a given sample It uses copper atrich bends with the peptide bonds in proteins under alkali ne conditions. This forms a moriovalere copper lon which can then recht with the Felin ciocalten reagent which is lum can be reduced into a blue colored Substance. This blue color can be measured using a sputrophotometer 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, Felin iccatt- reagent, two unknown solution, distilled acu re water, Reagent A, Reagent B PROCEDURE 1. First seven test tubes were cleaned and marked as blank, 31, 52, 53, 54, S, T, and T₂ (unknown solution) 2. Only distilled wooler was added to the blank tube, fortified by an alkaline copper sulfate solution and Folin-Worarteau reagent. 3. In the tubu marked as T, cy T₂, two unknown samples of protein which are of different concentratior. was adald followed by amp of distilled water 5 ml alkaline CuSOA scliction Folin-ciocalteau reagent and 50041 of 402,0 4,0 6,0 8 and 1.0 ml of the protein solution wore pipetted out from the tubes marked as 51,52 53. Sa eund so and it was made up to the volume 3ml by adding custilled water. 5. To earn tube ih ml of alkaline CuSO4 solution were Added, mixed well and allowed it to stand at sroom temperature for 10 minutes. 6. Then to each tube 0.5 ml of Folin ciocaltaen sreage m was added and mixed with the help of vortex 7 After vortexing, all the tubes were kept in a dark place for 20 minutes. 8. Then measured the absorbance of the purple blue Colour at 660nm. 9. Readings of eaun tube wire noted and a graph was plotled. 10 Protein conuntration of the given unknown sampl were calculated from the graph. RESULTS The protein concentration in the given unknown sample (Ti) is 74 ng/ml. The concentration of protein in the given unknown go sample (12) is 20 µg/ml