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

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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

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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