User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/04

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February 4th, 2015

Observations from overnight reactions...

All reactions involving the proteases proteinase K, trypsinItalic text, and chymotrypsin resulted in the formation of a purple colloidal solution from the cleavage of the solid phase AuNP fibers. All four pepsin reactions did not demonstrate any visible changes...yet again.

UV-Vis samples of all 16 reactions were run from 200-800 nm, with a large-volume quartz cuvette. The solutions were mixed before being added. The cuvette was washed twice with 0.5 M HCl, once with 1% SDS, and dried with Methanol.

Trypsin, Chymotrypsin, and Proteinase K all had discernible degradation of AuNP fibers at the conditions tested. The absorbance values were all relatively similar between the proteases. The maximum wavelengths were between 526 and 529 nm, which were smaller than expected. This means the colloid produced had smaller diameter particles. This will be confirmed with gel analysis.

Pepsin

Trypsin

Chymotrypsin

Proteinase K

Sample Preparation for Next Week

1. 0.00125 g of proteinase K was measured out and stored in the freezer to be used in the preparation of five proteinase K solutions next Tuesday.

  • Four 5 mL samples for Bradford and gel analysis (1 μM, 100 nM, 10 nM, 1 nM)
  • Two 2 mL cuvette samples for in situ UV-Vis kinetics analysis


2. 0.00105 g of lysozyme protein was dissolved in 10 mL of DI water to produce a 100 μg/mL lysozyme stock solution to be used in the production of a Bradford calibration curve next week.


3. 30 mL of SDS PAGE Sample buffer was prepared from the following:

  • 3.75mL 0.5M Tris pH 6.8 ---> 6.10737 g Tris in 100mL water
  • 15.0mL of 50% glycerol ---> 7.5mL glycerol + 7.5mL water (density of glycerol: 1.26 g/mL)
  • 0.3ml of 1% Bromphenol Blue ---> 0.00945 g Bromphenol Blue in 1mL water
  • 6.0mL 10% Sodium Dodecyl Sulfate (SDS) ---> 1.495 g SDS in 14.95 mL water
  • 4.95 mL of H2O

Protocol Preparation

Protocols for next week's Proteinase K reactions and analyses can be found in the entries for February 10th and 11th.





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