User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/03: Difference between revisions
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most of these also talked about adding an inhibitor to stop it... | most of these also talked about adding an inhibitor to stop it... | ||
==''' | =='''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 | |||
[[Image:1uM.Chymotrypsin.Kinetics.png]] | |||
[[Image:100nM.Chymotrypsin.Kinetics.png]] | |||
[[Image:10nM.Chymotrypsin.Kinetics.png]] | |||
[[Image:1nM.Chymotrypsin.Kinetics.png]] | |||
==''' | =='''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''' | |||
=='''CaCl<sub>2</sub> Buffer Preparation'''== | |||
A 50 mM Tris-HCl/ 10 mM CaCl<sub>2</sub> pH 8.155 buffer was prepared with 6.05923 g of Tris-HCl and 1.1098 g of CaCl<sub>2</sub> in a 1000 mL volumetric flask. Acid was added to a final pH of 8.155, however N/2 HCl acid contributed to a higher overall volume of 1040 mL. | |||
Final concentrations: | |||
[Tris-HCl]<sub>final</sub> = 48 mM | |||
[CaCl<sub>2</sub>]<sub>final</sub> = 9.6 mM | |||
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March 3rd, 2015Enzyme Condition NotesProteinase K
Trypsin
Chymotrypsin
OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl2 Thermolysin
Pepsin
most of these also talked about adding an inhibitor to stop it... Bradford Analysis of Trypsin Reaction Samples250 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.
1 nM Trypsin In Situ KineticsRan 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 CaCl2 Buffer PreparationA 50 mM Tris-HCl/ 10 mM CaCl2 pH 8.155 buffer was prepared with 6.05923 g of Tris-HCl and 1.1098 g of CaCl2 in a 1000 mL volumetric flask. Acid was added to a final pH of 8.155, however N/2 HCl acid contributed to a higher overall volume of 1040 mL. Final concentrations: [Tris-HCl]final = 48 mM [CaCl2]final = 9.6 mM |