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To determine the concentration of an unknown protein, a Bradford Assay was used.
To determine the concentration of an unknown protein, a Bradford Assay was performed. Materials needed included Bradford Reagent, bovine serum albumen (BSA), and protein (MBP). Standard solutions of BSA (1mL each) in water of 10, 8, 6, 4, 2, and 1ug/mL were prepared. First, 3mL of a 10ug/mL stock solution was created. From this stock solution the standards, as well as the sample of unknown protein, were made. The sample of the unknown protein should be 1000 times too concentrated. Furthermore, a blank for the Bradford Reagent and the protein were prepared. A UV-visible spectra was taken of each solution with a wavelength range of 200-800nm.
Standard amounts needed to make concentration curve
concentration BSA [micrograms/mL]
Chart 2: Is a graph of Absorbance vs. Wavelength for all of the concentrations.
Chart 3: Is a graph of molar absorbtivity vs. wavelength. This was found using the equation A=Ebc. Concentration of the of the protein blank/unknown was found by taking its absorbance at 595nm, and solvin for x from our calibration curve. We then obtained the value of 0.5321 ug/ml. This was then corrected by taking the original concentration (2ug/mL)=(0.53211yg/ml), so (0.53211ug/ml)(.1ml)=(x)(1ml) which is equal to 0.053217 ug/ml. Concentration of protein in water. The concentration of protein in water was then use to caculate E. (595nm)=(E)(0.01m)(00.05321). Therefore, E = 187.9310669 at 595nm.
Chart 4: Is a graph of Protein Absorbance in water vs. Wavelength. We can use the Absorbance of protein at 595nm to determine the molar absorbtivity, and eventually, find the concentration of protein in the unknown.
Calculations: Concentration of Unknown= 0.05 mg/ml E at 595nm of the protein in water = 187.93
What concentration do you calculate for your unknown? What does the absorbance of just the protein in water (no Bradford reagent) look like? What is the ε value for the protein at different wavelengths? Matt Hartings 21:48, 20 September 2011 (EDT)