User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/15
SDS Page Gel Electrophoresis
I. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
- The comb and tape was removed from each gel
- The wells of each gel were rinsed with the prepared running buffer dilution
II. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
- The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.
III. Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner:
- The samples were heated for 5 min at 100C in the thermocycler pre-loading
Gel AO
Well | Sample |
1 | Ladder |
2 | BLANK |
3 | 1 μM Proteinase K (I) |
4 | 100 nM Proteinase K (I) |
5 | 10 nM Proteinase K (I) |
6 | 1 nM Proteinase K (I) |
Gel AI
Well | Sample |
1 | Ladder |
2 | BLANK |
3 | 1 μM Proteinase K (II) |
4 | BLANK |
5 | 100 nM Proteinase K (II) |
6 | BLANK |
7 | 10 nM Proteinase K (II) |
8 | BLANK |
9 | 1 nM Proteinase K (II) |
Gel BI
Well | Sample |
1 | Ladder |
2 | BLANK |
3 | 1 μM Trypsin |
4 | 100 nM Trypsin |
5 | 10 nM Trypsin |
6 | 1 nM Trypsin |
7 | BLANK |
8 | 1 μM Chymotrypsin |
9 | 100 nM Chymotrypsin |
10 | 10 nM Chymotrypsin |
11 | 1 nM Chymotrypsin |
Gel BO
Well | Sample |
1 | Ladder |
2 | BLANK |
3 | 1 μM Thermolysin |
4 | SKIP |
5 | 100 nM Thermolysin |
6 | SKIP |
7 | 10 nM Thermolysin |
8 | BLANK |
9 | 1 nM Thermolysin |
IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W.
NOTE: The top of the chamber was not functioning properly in that a closed circuit was not established and the top had to be manually held/pushed down for the entirety of the run.
V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours
VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained:
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