User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/11
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In situ 1 µM Proteinase K Kinetics Run
To run the 1 nM (µM) concentration of Proteinase K and track over time for kinetics workup.
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.
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.
Proper Sample Prep
Bradford Analysis of Samples from Yesterday
Bradford analysis of the samples extracted yesterday will be run.
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.