User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/11

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February 11th, 2015

In situ Kinetics

Ocean Optics will be run at 37°C, scanning every 5 minutes for 6 hours.

The incorrect stock solution was used to make the protease solution. The concentration being tested is not 1 nM. It was calculated to be 1 µM for protease. This reaction will be re-run tomorrow with the proper enzyme concentration.

  • For 10 nM, take 69 µL of 0.4325 µL (10 µL stock, 990 µL buffer) protease, and dilute with 2.931 mL of buffer.
  • For 1 nM, take 6.9 µL of 0.4325 µL protease, and dilute with 2.993 mL of buffer.

1µM ProtK Kinetics Chart.png

1µM ProtK Kinetics AbsvsTime Chart.png

Bradford Analysis of Proteinase K 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 1.550 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.

Proteinase K Bradford 1uM.png

Proteinase K Bradford 100 nM.png

Proteinase K Bradford 10nM.png

Proteinase K Bradford 1nM.png