User:Benjamin Friedel/Notebook/CHEM 471/2015/09/30

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Objective

Today's objective was to complete the fluorescence analysis from yesterday (09/29/2015) as the blank standards were not run yet. Additionally, the objective was to analyze a-chymotrypsin degradation of lysozyme AuNP fibers over time at increased temperature (37˚C) through UV-Vis Bradford Analysis.

Protocol

Lysozyme Protease Degradation Fluorescence Analysis On 09/29/15 all of the lysozyme standards were analyzed, today the blank standard were analyzed using the same procedure.

Bradford Analysis of Protease Degradation

  1. First, a stock solution of our group's protease, alpha-chymotrypsin, was made in 1mL Tris/CaCl2 buffer (pH=8). The concentration of the protease was 40.625µM.
  2. Second, 7 samples of AuNP fibers (that Dr. Hartings synthesized earlier) were prepared for analysis using the following procedure
    1. centrifuge samples at 1500rpm for 1 min
    2. pipette supernatant off of samples (water)
    1. Samples were then incubated with protease for specific time points, after which Bradford analysis was conducted on them to monitor digestion with UV-Vis. The time points of incubation at 37˚C are as follows.
      1. 10 min
      2. 15 min
      3. 30 min
      4. 45 min
      5. 1 hour
      6. 1.5 hours
      7. 2 hours

Calculating Protease Volume in Reaction Mix

  1. To determine what volume of our 40.625uM protease stock solution to add to the reaction mix with the fibers to be incubated the equation M1V1=M2V2 was used with M1= molarity of protease stock, V1= volume of protease stock added to reaction mix, M2= molarity of protease in reaction mix, V2= volume of reaction mix
    1. The target protease concentration in the reaction mixes was 1uM and the target volume was 1mL, thus the volume of protease stock needed to be added to each reaction mix was 24.6ul.

Calculating Buffer Volume in Reaction Mix

  1. To determine what volume of Tris-CaCl2 buffer to add to the sample to bring the volume up to 1mL, the volume of protease to be added was subtracted from 1mL as it is assumed that most liquid was removed from the fiber samples after they were centrifuged.
    1. 1000ul total-24.6ul protease = 975.4ul buffer
    2. This volume of buffer was added to the spun down and supernatant-removed lysozyme AuNP sample tubes. Protease was not added until a moment before incubation commenced for an individual sample (10min incubation vs. 2 hr).

Blank Preparation

  1. Blanks were prepared to account for the absorbance of protease alone and protease self digestion
    1. For each timed sample a blank was prepared and treated in the same way.
    2. Blanks samples consisted of the same volume of protease and buffer as in the Lysozyme samples, however no lysozyme was added. This maintains the same protease concentration. Again, protease was not added until moments before incubation commenced and blanks were incubated for the same time and in the same conditions (rocking at 37˚C) as their respective lysozyme sample equivalents.

Bradford Analysis

  1. During sample and blank incubation, preparations were made to analyze those samples and blanks with Bradford reagent under UV-Vis.
  2. First, 600µL of pre-mixed Bradford dilution (1:3, bradford:buffer) into 14 plastic cuvettes (7 for the lysozyme samples, 7 for their respective blanks) for UV-Vis measurement.
  3. After each timed sample and blank was finished incubating for the above time points the following procedure was followed immediately.
    1. First, samples were removed from the water bath and centrifuged down at 300 RPM for 1 minute (according to previous year's lab notebooks, lower RPMs allow for less disturbance of the fibers)
    2. Then according to Dr. Harting's procedure, 1650uL of buffer was added to the cuvettes that would be used for the sample and blank analysis.
    3. Following addition of buffer, 750uL of the incubated sample reaction mix was added to its individual cuvette. Similarly, 750ul of blank reaction mix for that time point was added to its own individual cuvette with buffer.
  1. The UV-Vis spectrum was recorded for the sample and blank cuvettes from 400-800nm.
  2. These steps were repeated for the 10min, 15min, 30min, 45min, 60min, 90min and 120min samples.

Data

Figure 1: Lysozyme Degradation by alpha-Chymotrypsin Absorbance by Wavelength Figure 1 below shows the Uv-Vis absorbance spectra for each of the timed Bradford samples corrected for blank, background from 700-800nm and isosbestic point at 535nm. To correct for the blank the blank spectra for each corresponding incubation time was subtracted from the lysozyme sample spectra for that same incubation time. To correct for background, the average absorbance between 700-800nm was calculated for each blank corrected spectra and subtracted from that spectra. To correct for isosbestic point absorbance at 535nm for each of the blank and background corrected spectra was subtracted from each respective spectra so that they all are equal to zero absorbance at 535nm. Jnb09.30.15bradfordtimedlysozymeachymotrypsin.png

Figure 2: Lysozyme Degradation by 1uM alpha-Chymotrypsin Bradford Calibration Curve Figure 2 below shows the calibration curve of absorbance vs. protease incubation time in minutes and compares the samples to the blanks. For this data, the lysozyme sample spectra did not have their respective blank data subtracted from and were corrected for background and isosbestic point as shown above. Similarly, the blank data was corrected for background and isosbestic point as shown above. Jnb09.30.15bradford1uMcalibrationcurve.png