User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/25

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February 25th

1) Set up Chymotrypsin reactions

2) Bradford data work-up/analysis

3) Prep for SDS page/gel next week!

Chymotrypsin Reaction Preparation

0.00060 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [trypsin] = 24.00 μM


Bradford/Gel Analysis samples to be run:

1. 0.208 mL of stock solution in 4.792 mL of Tris/NaCl buffer

  • [chymotrypsin]final = 1 μM

2. 0.020(8) mL of stock solution in 4.979 mL of Tris/NaCl buffer

  • [chymotrypsin]final = 100 nM

3. 0.002(1) mL of stock solution in 4.998 mL of Tris/NaCl buffer

  • [chymotrypsin]final = 10 nM

4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.24 μM

    • Take 0.020(8) mL of dilution in 4.979 mL of Tris/NaCl buffer
  • [chymotrypsin]final = 1 nM

Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube. A 500 μL aliquot was obtained every 5 minutes from the 1 μM and 100 nM samples. Timepoints including: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min. A 500 μL aliquot was obtained every 30 minutes from the 10 nM and 1 nM samples. Timpoints including: 0 min, 30 min, 60 min, 90 min, 120 min, 20 hrs, 21 hrs, 22 hrs, 23 hrs. The samples were shaken at 236 rpm and 37 C for the duration of the reaction time.

1 µM Trypsin In Situ Kinetics

Protocol

90.7 µL of the stock Trypsin was diluted with 2.909 mL of Tris/NaCl for a final concentration of 1 µM. Fibers were spun yesterday and the liquid was extracted off the top. OceanOptics was run at 2 hours, scanning every 30 seconds.

Results

Workup is coming. will compare to 1 µM Proteinase K.

100 nM Trypsin In Situ Kinetics

Protocol

9.07 µL of the stock was diluted with 2.991 mL Tris/NaCl. Fibers were spun at 1500 RPM inadvertently. Same reaction conditions as above.

Results

Stock Solution Preparation

1. 50 mL of 2.5 mM HAuCl4 solution was made from 0.04361 g of HAuCl4 (MW 339.785 g/mol) in 50 mL of distilled H2O

2. 50 mL of 20 μM lysozyme solution was made from 0.01439 g of lysozyme (MW 14300 Da) in 50 mL of distilled H2O

AuNP Fiber Preparation

30 - 5 mL tubes of 45:1 [Au]:[Lysozme] AuNP solution were prepared with final concentrations of 0.25 mM and 5.56 μM of Au and lysozyme, respectively. Each tubed contained the following:

  • 0.5 mL of 2.5 mM HAuCl4
  • 1.39 mL of 20 μM lysozyme
  • 3.11 mL of distilled H2O

The AuNP solutions were placed in the oven at 80°C for four hours.