User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/10/27

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The objective of today was to run the Bradford Analysis of Protease Degradations for 1 nM Protinase K.


The protocol is identical to the one used on 9/29/15 except the fibers were spun down at 300 rpm for 10 minutes, not 1500 rpm for 1 minute.

  1. Prepping AuNP Fiber Samples
    1. 7 AuNP fiber samples were spun at 300 RPM for 10 minutes
    2. The supernatant was removed but the fibers were retained
    3. Protinase K tube 1C was mixed with 1 mL of 50 mM Tris and 10 mM CaCl2 pH 8 buffer
      1. Protinase K concentration: (0.00121g)*(1mol/28,900g)*(1/0.001L)= 0.0000419 M Protinase K
        1. Proteinase K dilution: (41900 nM)*(0.1 mL Proteinase K) = (x nM)*(10 mL total dilution) => x = 419 nM
        2. Proteinase K dilution: 0.1 mL of proteinase k solution was mixed with 9.9 mL of buffer
      2. Amount of Proteinase K solution needed for 1mL with 1 nM concentration: M1*V1 = M2*V2 => (419 nM)*(V1) = (1 nM)*(1 mL) => V1 = 0.0239 mL
      3. Amount of Buffer solution need to get to 1mL: (1mL total)-(0.0239 mL Protinase K solution) = 0.9761 mL buffer
  2. Incubating Samples
    1. 23.9 μL Protinase K and 976.1 μL buffer were mixed in the fiber tubes as well as blank eppendorf tubes
    2. Eppendorf tubes were incubated on a shaker in a 37°C water bath for 10 minutes, 15 minutes , 30 minutes, 45 minutes, 1 hour, 1.5 hours, and 2 hours for one of the fiber tubes and one of the blanks.
  3. Running Samples
    1. Once the time for a sample was up the tube was centrifuged at 300 rpm to collect the remaining fibers
    2. To a plastic cuvette was mixed in 600 uL of pre-mixed Bradford dilution, 750 uL of sample, and 1650 uL of buffer
    3. Samples were run from 400-800 nm on the UV-VIS


The graph below shows the absorbance of the samples as function of the wavelength of incident light when corrected. The six samples were first corrected for by subtracting the absorbance of the superblank of buffer and Bradford reagents. Then the samples were corrected for again by subtracting the results of the first correction by the blanks for their corresponding time periods. Then, the isosbestic point at 535 nm from each samples was subtracted from all the wavelengths of that sample. Bradford Assay of 10nM AuNP Fibers with Lysozyme Digestion, Wavelength vs. Absorbance at Various Times .png

The graph below shows the peak for the absorbance at 600 nm. For the first 90 minutes the absorbance is realtively stable at around 0.04 with dips at 15 minutes and 60 minutes. It is unknown if these dips are due to peptide concentration or human error. The absorbance then increases after 60 minutes. It would be desired to take additional samples past two hours to see how the additional time would affect the data.

AMS Bradford Assay of 10nM AuNP Fibers with Lysozyme Digestion, Absorbance at 600nm vs. Time.png