User:Khyra A. Neal/Notebook/Chem 571/2014/09/10: Difference between revisions

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==Data Analysis==
==Data Analysis==
*Calculating the purity of BSA stock solution
1. Purity of BSA stock solution
**
[[Image:BSAPurity.jpg]]
Based on the spectra above, the absorbance of BSA at 280 nm = 0.761
* Using Beer Lambert Law →A= Ε L C
** c=0.761/ 43824 = 1.736 X 10<sup>-5</sup>M
**Purity= [UV Measured] / [Mass Measured] *100
***1.736 X 10<sup>-5</sup>M / 1.5204 X 10<sup>-5</sup>M *100 = 114%
 
'''NOTE''' The extinction coefficient for BSA at 280 nm was found in literature to be 43,824. The percent purity of BSA was found to be greater than 1 possibly indicating that the polystyrene cuvettes weren't completely clean or BSA did not dissolve completely in solution and stuck to the cuvette.
 
*Because the Bradford reagent has peaks at 460 nm and 630 nm and the Bradford-protein complex has a peak near 600, there will be significant overlap. A difference spectra is constructed to make up for the difference and is shown below:
[[Image:BSADifferenceSpectra.jpg]]
 
 
 


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September 10, 2014

Bradford Assay

Prepare Stock Solutions

  • Prepare 50 mL of saline solution (0.9 wt-% NaCl)
    • 0.9 g→100 mL = 0.9 g/2= 0.45 g NaCl in 50 mL
    • Store in 45 mL falcon tube
  • Prepare 50 mL of 50 mM Tris base/ 50 mM NaCl solution
    • 50 mM →1000 mL 2.5mM → 50 mL
      • mass of tris= 2.5 mmol * 121.14 g/mol = 0.303 g Tris
      • mass of NaCl= 2.5 mmol * 58.44 g/mol = 0.1461 g NaCl
  • Prepare stock solution of BSA 5mg in 5 mL →1.01 μg/μL
    • (0.00505 g BSA /mol) * (1 mol/66,430 g) / 0.005 L = 1.5204 X 10-5M = 0.0152 mM

UV-Vis Analysis

Record UV-Vis Spectrum for stock solutions (BSA, Saline Solution, and Tris Buffer)

  • Make 7 standard solutions (1mL each) of 1 μg/μL, 4μg/μL, 6μg/μL, 8μg/μL, 12μg/μL, 16μg/μL, and 20μg/mL
    • Volume of stock solution is determined based on the concentration chart below.

Concentration (μg/μL) Amount of Bradford Reagent (μL) Amount of Tris Buffer (μL)
1 200 799
4 200 796
6 200 794
8 200 792
12 200 788
16 200 784
20 200 780


  • All concentrations were diluted to 1 mL total volume with Tris buffer solution and placed in 1.5 mL centrifuge tube.
  • Tubes were closed and vortexed for 5-10 seconds and sat for 5 minutes before running UV-VIs.
  • Record UV-Vis Spectrum for stock solutions (BSA, Saline Solution, and Tris Buffer) between 200 nm and 800 nm.
  • Record UV-Vis Spectrum for 7 standard solutions between 400 nm and 800 nm.
  • Make duplicate blanks for
    • 1 mL Tris/NaCl buffer
    • 200 μL Bradford reagent + 800 μL buffer
    • Record their UV-Vis spectra between 400 nm and 800 nm.

NOTE ALL UV-Vis spectra were run using polystyrene cuvettes. Solutions were discarded in waste bottle and polystyrene cuvettes were placed in a wash tube for cleaning.

Data Analysis

1. Purity of BSA stock solution Based on the spectra above, the absorbance of BSA at 280 nm = 0.761

  • Using Beer Lambert Law →A= Ε L C
    • c=0.761/ 43824 = 1.736 X 10-5M
    • Purity= [UV Measured] / [Mass Measured] *100
      • 1.736 X 10-5M / 1.5204 X 10-5M *100 = 114%

NOTE The extinction coefficient for BSA at 280 nm was found in literature to be 43,824. The percent purity of BSA was found to be greater than 1 possibly indicating that the polystyrene cuvettes weren't completely clean or BSA did not dissolve completely in solution and stuck to the cuvette.

  • Because the Bradford reagent has peaks at 460 nm and 630 nm and the Bradford-protein complex has a peak near 600, there will be significant overlap. A difference spectra is constructed to make up for the difference and is shown below:
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