User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/13

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Objective

To determine the concentration of an unknown sample of maltose binding protein (MBP).

Description

  1. Make standard solutions of 10, 8, 6, 4, 2, and 1 mg/mL of BSA from a stock solution with a concentration of 1.46 mg/mL.
  2. Mix each standard solution with 200 μL of Bradford reagent by vortexing.
  3. Dilute the unknown MBP solution to 10 μL of protein solution in 10 mL of water.
  4. Make two blanks, one of pure water and one of 800 μL MBP in 200 μL water, and mix by vortexing.
  5. Prepare a cuvette of 1 mL of unknown MBP solution.
  6. Take UV vis spectra (200 nm-800 nm) of all samples and create a standard curve.
  7. Use regression line to find the concentration of the unknown MBP solution.

Data

Calculations:

  • Determining the required concentration of BSA in M.
10 μg/mL × (1 g/1×106 μg) × (1000 mL/1 L) × (1 mol BSA/66776 g) = 1.50×10-7 M
  • Determining the volume of standard BSA solution required to make 1 mL of dilution.
M1 × V1 = M2 × V2
0.0146 mg/mL BSA × V1 = (10×10-3 mL)(1 mL)
V1 = 0.685 mL
  • Finding molar absorptivity
A = εbc
0.507 = ε(1cm)(1.50×10-7 M)
ε = 3.38×106 1/cmM


Results:

The absorbance values for the water and Bradford mixture were subtracted from those of the BSA and Bradford mixture to created corrected absorbance values that reflect the absorbance of the BSA only. Those values at 595 nm were plotted against concentration on a graph, and a line of best fit was determined.

The value for the absorbance of water and Bradford was also subtracted from MBP and Bradford. The value for the corrected MBP absorbance was plugged into the equation determined from the line of best fit in the Absorbance vs. Concentration graph [absorbance=0.0487(concentration)+0.0247]. The concentration of the unknown MBP was determined to be 1.074 mg/mL. Because the protein solution used in the experiment was diluted 1000x, the actual concentration of the protein sample is 1.074 mg/mL × 1000 = 1.074 g/mL. Converted to mol/L, the concentration of the unknown protein is 2.527×10-2M.


A second graph of Molar Absorptivity vs. Wavelength was created, to illustrate the changes in the amount of light absorbed per mole of as the wavelength was changed by the UV vis.



In addition, a graph illustrating the absorbance of all of the concentrations of BSA and of the MBP was prepared to compare the absorbance values of each.

Also, a graph showing the absorbance of MBP only against wavelength was also made.

Notes

The solutions of BSA and Bradford reagent visibly appeared to be darker the more concentrated they were.



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