User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/03

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Qualitative Analysis of Protease Activity

Objective

Continue the qualitative analysis of protease activity on AuNP fibers. Pepsin, Trypsin, Chymotrypsin, and Proteinase K will be tested.

Procedure

Stock Solutions

  1. Pepsin - 16 mg/4 mL -- 115.6 μL
  2. Trypsin - 10.77 mg/4 mL
  3. Chymotrypsin - 11.56 mg/4 mL
  4. Proteinase K - 13.37 mg/4 mL

Solutions Tested

  1. Pepsin
    1. 57.6 µM (2.5 mL buffer, 2.5 mL enzyme)
    2. 11.56 µM (4.5 mL buffer, 0.5 mL enzyme)
    3. 1.156 µM (4.95 mL buffer, 0.05 mL enzyme)
    4. 0.12 µM (5 mL buffer, 0.005 mL enzyme)
  2. Trypsin
    1. 57.6 µM (2.5 mL buffer, 2.5 mL enzyme)
    2. 11.56 µM (4.5 mL buffer, 0.5 mL enzyme)
    3. 1.156 µM (4.95 mL buffer, 0.05 mL enzyme)
    4. 0.12 µM (5 mL buffer, 0.005 mL enzyme)
  3. Chymotrypsin
    1. 57.6 µM (2.5 mL buffer, 2.5 mL enzyme)
    2. 11.56 µM (4.5 mL buffer, 0.5 mL enzyme)
    3. 1.156 µM (4.95 mL buffer, 0.05 mL enzyme)
    4. 0.12 µM (5 mL buffer, 0.005 mL enzyme)
  4. Proteinase K (K)
    1. 57.6 µM (2.5 mL buffer, 2.5 mL enzyme)
    2. 11.56 µM (4.5 mL buffer, 0.5 mL enzyme)
    3. 1.156 µM (4.95 mL buffer, 0.05 mL enzyme)
    4. 0.12 µM (5 mL buffer, 0.005 mL enzyme)

NB - Pepsin has an optimal pH of 1.5-2, so a pH 2.14, 5 mM Citrate, 0.2 M NaCl buffer was made for Pepsin use. That may affect the results.

Reaction Conditions

The AuNP fibers were spun down at 300 RPM for 4 minutes. The supernatant was removed, and the prepared solutions were added on top of the solid fibers. The tubes were placed in a shaker at 37°C. They will be analyzed tomorrow.

Results

Proteinase K, Trypsin, and Chymotrypsin all had visible color changes. Pepsin did not have a visible color change. UV-VIS analysis can be found on tomorrow's page.




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