User:Sydney Marshall/Notebook/Protease Project/2015/10/27

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Objective

  • Conduct Bradford Analysis between 10 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
      • (0.00121g)(1mol/28900g)(1/0.001L)=41868 nM Proteinase K
      • M1V1 = M2V2 => (41868 nM)*(V1) = (10nM)*(1mL) => V1 = 0.238843 uL
      • Dilution to 10nm
        • M1V1 = M2V2 => (41868 nM)*(0.1mL) = (x)*(10mL) => M2 = 418.685 nm
        • M1V1 = M2V2 => (418.68 nM)*(V1) = (10nM)*(1mL) => V1 = 23.9 uL (976.1 uL of buffer needed 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

Bradford Assay of 10nM AuNP Fibers with Lysozyme Digestion, Wavelength vs. Absorbance at Various Times .png
This graph shows the absorbance of the 10nm Proteinase K samples from 400nm to 800nm. These are corrected with their blanks. AMS Bradford Assay of 10nM AuNP Fibers with Lysozyme Digestion, Absorbance at 600nm vs. Time.png
This is the peak absorption vs time curve of 10nm Proteinase K digestion.