User:Jamie Nunziata/Notebook/Protease Research/2015/11/04

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Objective

The purpose of this lab is to use Bradford Assay to measure AuNP fiber degradation from a solution of 1nM a-chymotrypsin protease

Procedure

7 samples of AuNP fibers were spun down at 300rpm for 10 minutes.These fibers will be used for our 23 hours, 120min, 90min, 60min, and 30min samples. A 1:200 dilution of our stock protease was made in Tris buffer. We pipetted 5µL of our 43.539µM stock solution of a-chymotrypsin into 995µL of Tris buffer. This brought the final concentration of the a-chymotrypsin to 0.20506µM. 4.88µL of the diluted a-chymotrypsin (in Tris) and 995.12µL of Tris buffer was added to each sample tube and a blank Eppindorf tube (no fibers), making the final solution 1nM 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_11_4_2015_CorrectedAborbanceSamples_Bradford.jpg

Image:JMN_11_4_2015_BlankSample600nm_Bradford.jpg

Image:JMN_11_4_2015_Sample600nm_Bradford.jpg .



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