User:Jamie Nunziata/Notebook/Protease Research/2015/10/27

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Objective

The purpose of this lab is to use Bradford Assay to measure AuNP fiber degradation from a solution of 10nM a-chymotrypsin protease

Procedure

7 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, 45min, 30min, 15min, and 10min samples. A 1:20 dilution of our stock protease was made in Tris buffer. We pipetted 50µL of our 43.539µM stock solution of a-chymotrypsin into 950µL of Tris buffer. This brought the final concentration of the a-chymotrypsin to 2.177µM. 4.59µL of the diluted a-chymotrypsin (in Tris) and 995.41µL of Tris buffer was added to each sample tube and a blank Eppindorf tube (no fibers), making the final solution 10nM a-chymotrypsin.


To each cuvette, the following was added:

  • 1650µL of Tris buffer
  • 750µL of incubated 1µM sample (or blank)
  • 600µL of diluted Bradford Assay in Tris buffer (1:3)

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

Absorbance for each cuvette was measure via UV-Vis between the emission sprectrum of 400-800nm, and the data is recorded below

Data

Image:JMN_10_27_2015_CorrectedAborbanceSamples_Bradford.jpg

Image:JMN_10_27_2015_BlankSample600nm_Bradford.jpg

Image:JMN_10_27_2015_Sample600nm_Bradford.jpg



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