User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/24

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February 24th

TO DO:

1) Bradford of all Trypsin samples.

2) Start on in-situ kinetics for trypsin.

3) Calculations for Chymotrypsin - prep for Wednesday.

Bradford Analysis of Trypsin Reaction Samples

250 μL of each protein sample at every time point (collected yesterday) was combined with 200 μL of Bradford reagent (1:4 Bradford:Tris/NaCl), and 550 mL of Tris/NaCl buffer and run from 400 - 800 nm on the UV/Vis spectrophotometer. The following are the results with the blank subtracted out and zeroed at a wavelength of 535 nm.

Trypsin Bradford 1uM.png

Trypsin Bradford 100 nM.png

Trypsin Bradford 10nM.png

Trypsin Bradford 1nM.png

In-situ kinetics for 10 nM trypsin

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