п»їExperiment two: Estimation of protein attention
Name: Wong Jiun Hao
Module: Biological Science
Table of Contents
Table of Contents2
The concentration of protein within a sample can be obtained by using the Liverpool protein assay method. The method requires a spectrophotometer and it is easily operated. The Bradford necessary protein assay is known as a colorimetic assay based on the interaction in the blue dye " CoomassieвЂќ and the healthy proteins. The blue ye firsts donates free electron for the protein to result in the proteins to lose it is native state and revealing it hydrophobic pockets. These hydrophobic pouches then binds to the Coomassie dye through Van Dieser Wall forces. The holding protein in that case stabilizes the blue sort of the Coomassie dye. Consequently, the absorbance reading can be acquired and used to measure the protein concentration which is the amount of necessary protein complex within the solution. To find the protein attention from the standard curve. 1st, draw the line of best fit and include the equation into the graph. The R2 will be as near to 1 as possible. Apply the Beer Lambert Law A= О»bc where a is the tested absorbance, О» is the slope of the common curve, n is the entire cuvette, and c is a concentration staying determined.
1 . To prepare a standard curve of absorbance versus protein attentiveness by using Boeotian Serum Albumin (BSA). installment payments on your To determine BSA concentration in two test solutions.
3. To determine proteins concentration in apple juice.
several test pontoons were prepared and marked. The appropriate quantity of BSA and unadulterated water were pipetted in the respective test out tubes in accordance Table a few. 1 . 500Вµl of Branford Reagent had been added in to all the check tubes. Test tubes were all vortexed and blended. The alternatives were remaining for two minutes plus the absorbance within just 60 minutes at a wavelength of 595nm was written. The standard competition of absorbance versus attention of BSA was drawn. Part 2
Two sample alternatives A and B were given with not known BSA focus. The two samples were pipetted into evaluation tubes correspondingly. Distilled water and Branford reagent was added according to Stand 3. installment payments on your The test pipes were vortexed and blended. The absorbance at wavelength 595nm was recorded. The BSA concentration of samples A and W were based on using the regular curve used Part 1 ) Part a few
A sample solution of apple juice in tube C was given. Test C was pipetted into a test pipe together with distilled water and Branford reagent according to Table three or more. 2 . The sample was vortexed and mixed. The absorbance at wavelength 595nm was scored and registered. The necessary protein concentration of sample C was determined using the standard curve via Part 1 ) The sample was diluted if the absorbance reading beyond range in the standard shape. The results were compared with the protein concentration stated in the nutritional information of the apple juice shown in Figure three or more. 1 .
Stand 3. one particular: Volume of Distilled water and Branford reagent, concentration of BSA, and Absorbance of sample in 595nm
Concentration of BSA(Вµg/ml)
Absorbance in 595nm
a couple of
1 ) 05
1 . 67
2 . 35
Desk 3. 2: Volume of Test solution, Unadulterated water and Branford reagent and Absorbance of test at 595 nm. Sample
Absorbance in 595nm
References: 1 . http://www.sigmaaldrich.com/life-science/proteomics/protein-quantitation/bradford-reagent.html
2 . Bradford protein assay. (2014, March 28). InВ Wikipedia, The Free of charge Encyclopedia. Recovered 01: twenty nine, November seventeen, 2014, fromВ http://en.wikipedia.org/w/index.php?title=Bradford_protein_assay&oldid=631530871