User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/03

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March 3rd, 2015

Enzyme Condition Notes

Proteinase K

  • MW: 28,900 Da
  • optimal pH: 8.0
  • optimal temperature range: 20-60C
  • optimal concentration range: 1.73 - 3.46 uM
  • Ca is a stabilizer especially at low concentrations (1-5 uM)
  • suggested buffer: 50 mM Tris-HCl (pH 7.5) 5 mM CaCl2

Trypsin

  • MW: 23,300 Da
  • optimal pH: 7.5-8.5
  • optimal temperature range:
  • suggested buffer: 46 mM Tris-HCl (pH 8.1) 11.5 mM CaCl2

Chymotrypsin

  • MW: 25,000 Da
  • optimal pH: 7.8-8.0
  • 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2

OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl2

Thermolysin

  • MW: 36,200 Da
  • optimal pH: 8.0
  • recommended enzyme:protein ratios of 1:20 - 1:50
  • digestion buffer: 50 mM Tris-HCl (pH 8.0) 0.5 mM CaCl2

Pepsin

  • MW: 34,600 Da
  • optimal pH: 1.0-3.0
  • recommended enzyme:protein ratios of 1:20 - 1:100

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

1uM.Chymotrypsin.Kinetics.png

100nM.Chymotrypsin.Kinetics.png

10nM.Chymotrypsin.Kinetics.png

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

1nM Trypsin AbsvsTime Chart.png

1nM Trypsin Kinetics Chart.png

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.

Final concentrations:

[Tris-HCl]final = 48 mM [CaCl2]final = 9.6 mM