User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/11

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In situ 1 µM Proteinase K Kinetics Run

Objectives

To run the 1 nM (µM) concentration of Proteinase K and track over time for kinetics workup.

Protocol

Take 0.046 mL of dilution from step 4 above in 1.954 mL of Tris/NaCl buffer for a final [proteinase K]final = 10 nM. From there, dilute by a factor of 10 to decrease the final concentration to 1 nM. Ocean Optics will be run at 37°C, scanning every 5 minutes for 6 hours.

Results

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 of protease. This reaction will be rerun tomorrow with the proper enzyme concentration.

1µM ProtK Kinetics Chart.png

1µM ProtK Kinetics AbsvsTime Chart.png

Proper Sample Prep

  1. 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.
  2. For 1 nM, take 6.9 µL of 0.4325 µL protease, and dilute with 2.993 mL of buffer.

Bradford Analysis of Samples from Yesterday

Objectives

Bradford analysis of the samples extracted yesterday will be run.

Protocol

A 1 in 4 Bradford assay was made (1 mL Bradford in 3 mL of Tris/NaCl buffer). 250 μL of each protein sample at every time point (collected yesterday) was combined with 200 μL of Bradford reagent, 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.

Results

Proteinase K Bradford 1uM.png

Proteinase K Bradford 100 nM.png

Proteinase K Bradford 10nM.png

Proteinase K Bradford 1nM.png