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Protein Quantitation - Lab Report Example

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Bradford Protein Concentration Assay Name: Institution: Instructor: Course: Date: Abstract The measurement of protein concentrations in aqueous samples is an important assay in biochemistry research and development laboratories for applications such as enzymatic studies and also in providing data for biopharmaceutical lot release…
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Protein Quantitation
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Download file to see previous pages The following is a report on an experiment conducted to determine protein concentration of unknown samples using this method. Bradford Protein Concentration Assay Accurate protein quantitation is paramount to all experiments that are related to proteins in a lot of research topics in molecular biology, developmental biology, cell biology, neuroscience, and biochemistry. Different techniques have been developed to quantitate proteins in the last century, both for the total protein content and a single protein. Total protein content quantitation methods include Bradford assays. Bradford assay, which was initially described by Dr. Marion Bradford in 1976, is one of the commonly used methods to determine protein concentration. This method relies on formation of a complex between proteins in solution and the Coomassie brilliant blue G-250 dye. This dye exists in four different ionic forms. The more anionic blue form binds to proteins and has an absorbance at 590 nm. Protein concentrations can be known by determining the amount of dye in the blue ionic form, and by measuring the absorbance of the solution at 595 nm using a spectrophotometer (Becker, Caldwell & Zachgo, 1996). This dye binds mostly to arginine, tryptophan, tyrosine, histidine, and phenylalanine residues of the protein Materials and Reagents Protein standard: 1mg/mL Albumin Bradford reagent Distilled water Test samples A, B and C (Unknown protein) One 96-well plate Procedure First, the albumin standard solutions were prepared as follows: Concentration Albumin Distilled Water 0% 0ul 100ul 25% 25ul 75ul 50% 50ul 50ul 75% 75ul 25ul 100% 100ul 0ul Then the Bradford reagents were diluted with 300 ul (Bradford): 1500ul (Distill Water) (1:5 ratio). In the first trial, 180ul of the diluted Bradford reagents was added into the 96 well plate. Then 0%, 25%, 50%, 75%, 100% and Sample A, B, C, each 5ul was added to each well hole which contained 180ul of diluted Bradford reagent. These were tested with spectrophotometer and the results recorded. The experiment was repeated again the same way. In addition, Sample C was diluted with 3 different ratios as follows: 1:9 (Sample C: Distill Water) 1:99 (Sample C: Distill Water) 5:95 (Sample C: Distill Water) The results were recorded after the solutions were tested with a spectrophotometer. A standard curve of absorbance versus concentration protein was drawn. Results The results were recorded as follows: First Trial Results: 0% 25% 50% 75% 100% Sample A Sample B Sample C 0.092 0.145 0.161 0.169 0.162 0.095 0.161 0.763 Second Trial Results: 0% 25% 50% 75% 100% Sample A Sample B Sample C Sample C (1:9) Sample C (1:99) Sample C (5:95) 0.113 0.188 0.218 0.217 0.210 0.116 0.200 0.670 0.244 0.118 0.152 The responses of the standards were used to plot a standard curve. Absorbance values of unknown samples were then interpolated onto the plot for the standard curve to determine their concentrations as shown in the graph below. Discussion From the graph above, the optimum measurement wavelength for this assay is on sample C. Thus sample C has the highest protein concentration. Sample A has the same absorbance as 0% albumin and Sample B has the same absorbance as 50% albumin. It can therefore be concluded that sample A and Sample B have 0% and 50% protein concentra ...Download file to see next pagesRead More
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