User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/24

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10 nM Trypsin Kinetics Run

Objective

Run kinetics with 10 nM Trypsin.

Protocol

OceanOptics set to the same parameters (1 ms integration time, 10 scans/average, 37°C, scan every 5 minutes for 6 hours).

Trypsin was prepared from the 1:100 diluted stock. 90.7 µL of the trypsin was diluted to 3 mL with 2.909 mL of Tris/NaCl. Fibers were spun down and the liquid was extracted off the top. Both were added to the cuvette and the reaction was started.

Results

There was solid fibrous aggregate when the reaction finished.

10nM Trypsin AbsvsTime Feb 24.png

10nM Trypsin Kinetics Feb 24.png


Bradford Analysis of Trypsin Activity

Monika and Beatriz prepared the samples for Bradford analysis. A quartz 1 mL cuvette was used to see if aberrant results were due to cuvette or dilution issues. 250 μL of each protein sample was combined with 200 μL of Bradford reagent (1:4 Bradford:Tris/NaCl), and 550 mL of Tris/NaCl buffer and scanned from 400 - 800 nm.

Results

These are the corrected results:

Trypsin Bradford 1uM.png

Trypsin Bradford 100 nM.png

Trypsin Bradford 10nM.png

Trypsin Bradford 1nM.png