User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/11/11

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Objective

To analyze the activity of proteinase k at 1 nM using fluorescence

Description

The guide for analyzing a protease with fluorescence can be found here at Dr. Hartings notebook

  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 11 was mixed with 1 mL of 1 mL of 50 mM pH 8 phosphate buffer
      1. Protinase K concentration: (___)*(1mol/28,900g)*(1/0.001L)= 0.0000___ M Protinase K
      2. Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (____ μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.00__ mL
      3. Amount of Buffer solution need to get to 1mL: (1mL total)-(0.____ mL Protinase K solution) = 0.____ mL buffer

NEED TO GET DATA AS WELL AS DILUTION

  1. Incubating Samples
    1. ____ μL Protinase K and ____ μL buffer were mixed in the 7 fiber tubes as well as 7 blank eppendorf tubes
    2. Eppendorf tubes were incubated on a shaker in a 37°C water bath for 15 minutes, 45 minutes, 75 minutes, 45 minutes, 105 minutes, 135 minutes, 1220 minutes and 1440 minutes for one of the fiber tubes and one of the blanks.
  2. Running Samples
    1. Once the time for a sample was up the tube was centrifuged at 12000 rpm for 1 minute to collect the remaining fibers
    2. From either the sample or blank, 20uL was taken and placed in a 600uL eppendorf tube
      1. Added 140uL of Assay Buffer
      2. Added 40uL of Assay Reagent
    3. Taking Measurement
      1. Excitation at 390 nm
      2. Emission from 400 to 650 nm
      3. Both slit widths set to 10 nm
      4. Scan Speed of 200

Results

The image below shows the intensity for the fluorescence of samples and blanks from 400 to 650 nm.

To find the concentrations of peptide released at the various time intervals the samples and blanks had the area of their intensity integrated from 420 nm to 650 nm. The equation (Wavelength2 - Wavelength1)/2*0.5 was used and the sums of each interval totaled. The samples were then corrected for by subtracting the total area of the blank from the total area of the sample. The resulting intensities were then used to calculate the concentrations based off the calibration curve made on 10/07/15. The graph below shows the concentrations of the peptides as a function of time.

That so many early time data points are negative may either indicate that there is so little protein cleaved that background noise is affecting the results or indicate human error. That, combined with the massive spike at 1220 minutes, indicates that this data should be re-run at a later date.


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