User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/11: Difference between revisions
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'''Protocol''' | '''Protocol''' | ||
A 1 in 4 Bradford assay was made (1 mL Bradford in 3 mL of Tris/NaCl buffer). | 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''' | |||
[[Image:Proteinase_K_Bradford_1uM.png|500px]] | |||
[[Image:Proteinase_K_Bradford_100_nM.png|500px]] | |||
[[Image:Proteinase_K_Bradford_10nM.png|500px]] | |||
[[Image:Proteinase_K_Bradford_1nM.png|500px]] | |||
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Revision as of 08:41, 25 February 2015
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In situ 1 µM Proteinase K Kinetics RunObjectives 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.
Proper Sample Prep
Bradford Analysis of Samples from YesterdayObjectives 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
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