User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/30: Difference between revisions

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===Brasford Analysis of Protease Degradation===
===Brasford Analysis of Protease Degradation===
# First, we made a stock solution of our protease, alpha-chymotrypsin, using Tris/CaCl<sub>2</sub> buffer and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 40.625µM.
# Next, we prepared 7 samples of AuNP fibers that Dr. Hartings prepared for us:
## We spun the samples down at 1500 RPM for 1 minute
## We pipetted the water off of the samples
## We labeled the samples based on the time that they would be incubated in the 37 degree Celsius water bath:
### 2 hours
### 1.5 hours
### 1 hour
### 45 min
### 30 min
### 15 min
### 10 min
## We measured the mass of each of the samples (the mass included the mass of the Eppindorff tube and the sample inside it)
## Next, we determined how much of our protease stock and how much Tris/CaCl<sub>2</sub> buffer we should add to each of the sample tubes in order to make the final concentration of alpha-chymotrypsin 1µM. We did the following calculation:<br>Let<br>M<sub>1</sub>=concentration of protease stock = 40.625µM<br>V<sub>1</sub>=volume of protease stock needed<br>M<sub>2</sub>=concentration of alpha-chymotrypsin in the sample tube = 1µM<br>V<sub>2</sub>=volume of the solution in the sample tube = 1mL<br>M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub><br>V<sub>1</sub>=(M<sub>2</sub>V<sub>2</sub>)/(M<sub>1</sub>)<br>V<sub>1</sub>=((1µM)(1mL))/(40.625µM)<br><b>V<sub>1</sub>=0.0246mL=24.6µL</b>


==Data and Analysis==
==Data and Analysis==

Revision as of 09:07, 6 October 2015

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Objective

The purpose of today's lab work is to:

  1. Finish taking fluorescence data from yesterday's lab
  2. Use a Bradford Assay in order to measure alpha-chymotrypsin degradation as a function of incubation time

Protocol

Dr. Hartings's protocol for today is here. We are following his protocols for fluorescence and for the Bradford Assay.

Fluorescence Analysis of Protease Degradation

Yesterday, we measured the fluorescence of all of our samples of alpha-chymotrypsin + lysozyme. Today, we finished the fluorescence measurements for the alpha-chymotrypsin blanks as follows:

  1. We had finished creating our blanks yesterday. Just before we measured the fluorescence of each blank, we combined the following 1.5mL Eppindorf tubes:
    1. 20uL of the blank to be measured
    2. 140uL of Assay Buffer
    3. 40uL of Assay Reagent
  2. We then pipetted this mixture into a 1mL quartz cuvette and took fluorescence measurements (excitation wavelength was 390nm and emission wavelengths were 400 to 650nm).

Brasford Analysis of Protease Degradation

  1. First, we made a stock solution of our protease, alpha-chymotrypsin, using Tris/CaCl2 buffer and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 40.625µM.
  2. Next, we prepared 7 samples of AuNP fibers that Dr. Hartings prepared for us:
    1. We spun the samples down at 1500 RPM for 1 minute
    2. We pipetted the water off of the samples
    3. We labeled the samples based on the time that they would be incubated in the 37 degree Celsius water bath:
      1. 2 hours
      2. 1.5 hours
      3. 1 hour
      4. 45 min
      5. 30 min
      6. 15 min
      7. 10 min
    4. We measured the mass of each of the samples (the mass included the mass of the Eppindorff tube and the sample inside it)
    5. Next, we determined how much of our protease stock and how much Tris/CaCl2 buffer we should add to each of the sample tubes in order to make the final concentration of alpha-chymotrypsin 1µM. We did the following calculation:
      Let
      M1=concentration of protease stock = 40.625µM
      V1=volume of protease stock needed
      M2=concentration of alpha-chymotrypsin in the sample tube = 1µM
      V2=volume of the solution in the sample tube = 1mL
      M1V1=M2V2
      V1=(M2V2)/(M1)
      V1=((1µM)(1mL))/(40.625µM)
      V1=0.0246mL=24.6µL

Data and Analysis

Fluorescence

Figure 1: Fluorescence of Lysozyme in Varying Concentrations of alpha-Chymotrypsin (nM) as a Function of the Wavelength of Incident Light (nm)

The above figure shows the fluorescence of lysozyme in samples of varying concentrations of alpha-chymotrypsin as a function of the wavelength of incident light. The fluorescence of each sample was corrected for the blanks by subtracting the fluorescence of the blank from the fluorescence of the sample for every wavelength measured. Each curve on the graph represents a different concentration of alpha-chymotrypsin.

Figure 2: Fluorescence Intensity of Lysozyme as a Function of the Concentration of alpha-Chymotrypsin

The above image shows the fluorescence intensity of lysozyme as a function of the concentration of alpha-chymotrypsin that it is reacted with.

Bradford Assay


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