User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/04/14

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April 14th, 2015

SDS-PAGE Preparation

Solutions and samples were prepared for SDS-PAGE analysis (to be run tomorrow).

Sample Prep:

We prepared non-dilute samples (10 µL reaction sample/10 µL running buffer) for SDS-PAGE analysis. Here are the solutions we used for each enzyme concentration:

  1. Proteinase K Sample 1:
    1. 1 µM, 2 hrs
    2. 100 nM, 2.5 hrs
    3. 10 nM, 2 hrs
    4. 1 nM, 2 hrs
  2. Proteinase K Sample 2:
    1. 1 µM, modified protocol, 60 min
    2. 100 nM, modified, 80 min
    3. 10 nM, modified, 22 hrs
    4. 1 nM, modified, 21 hrs
  3. Thermolysin:
    1. 1 µM, 90 min
    2. 100 nM, 30 min
    3. 10 nM, 60 min
    4. 1 nM, 10 min
  4. Chymotrypsin:
    1. 1 µM, 1 hr
    2. 100 nM, 25 min
    3. 10 nM, 20 hrs
    4. 1 nM, 23 hrs
  5. Trypsin:
    1. 1 µM, 2 hrs
    2. 100 nM, 2.5 hrs
    3. 10 nM, 2 hrs
    4. 1 nM, 2 hrs

Solutions

  • 500 mL Fixative Solution (40% methanol, 10% acetic acid, 50% water)
  • 500 mL Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water)
  • 500 mL Destain Solution (10% acetic acid, 90% water)
  • 200 mL of 10X SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10 in 2000 mL of water