User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/03/03

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1 nM Trypsin In Situ Kinetics

Ran the final Trypsin In Situ reaction.

Protocol

9.07 µL of the diluted stock was added to 2.991 mL of Tris/NaCl buffer. Fibers were spun at 300 RPM for 20 minutes (the fibers did not sediment after the first 10 minutes), and the liquid was extracted. 2 mL of the Trypsin was added to the cuvette with minimum amount of liquid from AuNP fibers. OceanOptics was run at 37°C, spinning at 1000 RPM, scanning every 5 minutes for 6 hours total.

Results

There was barely a change in absorbance with this concentration of Trypsin.

Bradford Analysis of Trypsin Reaction Samples

250 μL of each protein sample at every time point 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.

  • Kinetics of Chymotropsin at 1μM, 100nM, 10nM and 1nM



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