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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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
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Fig 1. Fluorescence intensities of a) tyrosine b)Tryptophan at and c) Thioflavin T at 25 0c and three different pH values. The fluorescence intensities for these three were measured at 303, 348, 482 nm respectively, in all the experiments.
There was slight increase in Tryptophan fluorescence at pH 5.0 and 7.0.
Consequently, a destabilized protein level in the body, through inappropriate nutrition or through effects of diseases adversely affects body processes. The study of protein concentration in the body, as well as in substances is therefore fundamental to nutritionists and health care professionals.
This report examined the features of E coli in respect to the expression systems with an emphasis on the limitation number that have been addressed. For a high gene dosage to be achieved, the heterologous cDNAs are normally cloned into replicate plasmids in a fashion that is relaxed and are normally existence at 15-60 copies per cell.
he template strand of DNA, which contains a nucleotide sequence encoding for the protein identified in the answer to question 3c from the Transcription activity above:
Glycine, methionine, valine, glutamic acid, glutamine, cycteine, cysteine, alanine, serine, valine, cysteine,
With respect to their structure, biochemists often refer to four different aspects:
Secondary structure, local interactions held together by hydrogen bonds between the lone pair of electrons of an oxygen atom and the hydrogen attached to a nitrogen atom.
4. If a particular protein was absent, it would lead to errors in protein synthesis. Errors in protein synthesis disrupt cellular fitness, cause disease phenotypes, leads to loss of function of the protein (non-functional proteins) and protein misfolding.
After the 30 minutes of incubation the rank is removed from the water bath and the samples allowed to cool.1 ml of each reaction is transfered to a 1 ml cuvette and A562 in a spectrophotometer read. The spectrophotometer zero is set to
Proteomic is generally defined as the direct analysis of proteins in terms of their presence and relative abundance. Gel electrophoresis is a significant methodology employed for extraction of proteins in proteome analysis. The most commonly used technique in gel electrophoresis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
5 Pages(1250 words)Lab Report
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