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