User:Sydney Marshall/Notebook/Protease Project/2015/10/13

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Objective

  • Conduct Bradford Analysis between 100 nM Proteinase K and AuNP fibers

Description

The detailed protocol can be found in this notebook entry by Dr. Hartings.

  • AuNP Fibers Preparation
    • 7 eppendorf tubes with AuNP were centrifuged at 1500RPM for 1 min.
    • Their supernatant was aspirated
  • Protinase K 1 μM Preparation
    • Calculations
      • (0.00127g)(1mol/28900g)(1/0.001L)=44.000 μM Proteinase K
      • M1V1 = M2V2 => (44.000 μM)*(V1) = (1μM)*(1mL) => V1 = 0.00227mL = 2.3 μl Proteinase K (997.7 μl of buffer needed to add to make 1mL).
  • Incubation
    • 14 samples (7 with fibers, 7 blanks without fibers) were incubated in a 37°C shaking water bath for 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hrs, 1.5 hrs, and 2 hrs each.
  • Bradford Analysis
    • At each time point, the samples were taken out of the water bath and centrifuged at 13500 RPM for 1 minute.
    • 750 μL of each sample and blank was placed in a plastic cuvette with 600 μL of pre-mixed Bradford dilution and 1650 μL of buffer.
    • Samples were run from 400nm to 800nm on UV-Vis.

Results


The graph above is the samples corrected by their blanks for each time points.
This graph shows the peak absorption of the samples against each time point at 600nm. The relationship between absorption and time is non-linear.


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