User:Jamie Nunziata/Notebook/Protease Research/2015/11/10

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Objective

The objective of today's lab was to use a Fluorescence Assay to determine the rate a 1nM solution of a-chymotrypsin degrades AuNP fiber samples.

Procedure

6 samples of AuNP fibers were spun down at 300rpm for 10 minutes. These fibers will be used for our 48 hours, 24 hours, 120min, 90min, 60min, and 30min samples. A 1:200 dilution of our stock protease was made in phosphate buffer. We pipetted 5µL of our 51.172µM stock solution of a-chymotrypsin into 995µL phosphate buffer. 3.91µL of diluted a-chymotrypsin (in phosphate buffer) and 996.1µL of phosphate buffer was added to each sample tube and a blank Eppindorf tube (no fibers), making a final solution of 1nM a-chymotrypsin.


To each cuvette, the following was added:

  • 20uL of the blank or sample
  • 140uL of Assay Buffer
  • 40uL of Assay Reagent


A full lab procedure can be found in Nicole Bonan's notebook entry for today


Fluorescence for each sample was measured between the emission spectrum from 400 to 600nm, and the data is recorded below

Data

Image:JMN_11_10_2015_BlankSample_Fluorescence.jpg

Image:JMN_11_10_2015_SampleSlope_Fluorescence.jpg



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