User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/03
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March 3rd, 2015
Enzyme Condition Notes
OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl2
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.
1 nM Trypsin In Situ Kinetics
Ran the final Trypsin In Situ reaction.
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.
There was barely a change in absorbance with this concentration of Trypsin.
CaCl2 Buffer Preparation
A 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.
[Tris-HCl]final = 48 mM [CaCl2]final = 9.6 mM