User:Klare Lazor/Notebook/Chem-496-001/2011/09/14

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Objective

To determine the concentration of an unknown protein, a Bradford Assay was used.

Description

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.

Data

Standard amounts needed to make concentration curve

  • 1) 0.8ml of BSA Stock + 0.2mL of B.Reagent
  • 2) 0.6ml of BSA Stock + 0.2mL of B. Reagent
  • 3) 0.4ml of BSA Stock + 0.2mL of B. Reagent
  • 4) 0.3mL of BSA Stock + 0.2ml of B. Reagent
  • 5) 0.2ml of BSA Stock + 0.2mL of B.Reagent
  • 6) 0.1mL of BSA Stock + 0.2mL of B. Reagent
  • Water was than added to each dilution to make volume 10mL

concentration BSA [micrograms/mL]

  • 1)7.976
  • 2)5.982
  • 3)3.988
  • 4)2.991
  • 5)1.994
  • 6) 0.997

Blanks

  • 200ulBR + 8uL water
  • 200ulprotein + 8uL water

Graphs

Image:001final update .png


Image:000001 i lie final update.png


Chart 1: Is a graph of Absorbance vs. Concentration at 595nm. From this we can use the line of best fit to determine the concentration of protein in the unknown. We eliminated one data point because it was irrelevant. In addition, the line of best fit was set with a 0 intercept to get a better line of fit. y=.218x.

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)

Notes

Observations:

  • It was observed that the color of the Bradford Reagent upon addition to the protein was blue, and slowly, turned back to red when the concentration of the protein decreased.


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