User:Beatriz Gimenez De C./Notebook/572/2015/04/08
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Tasks
- Perform SDS Page Gel Electrophoresis with solutions made on Tuesday
- A 10x SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10.
- 100 mL of the 10x buffer was added to a 1000 mL volumetric flask
- load on wells 20 μL of each of the samples prepared yesterday, headed for 5 min on thermocylender + a ladder/marker in the following order:
- A 10x SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10.
Gel AO
Well | Sample |
1 | Ladder |
2 | 1 μM Proteinase K - Dilute |
3 | 100 nM Proteinase K - Dilute |
4 | 10 nM Proteinase K - Dilute |
5 | 1 nM Proteinase K - Dilute |
6 | Blank |
7 | 1 μM Proteinase K |
8 | 100 nM Proteinase K |
9 | 10 nM Proteinase K |
10 | 1 nM Proteinase K |
Gel AI
Well | Sample |
1 | Ladder |
2 | Blank |
3 | 1 μM Trypsin - Dilute |
4 | 100 nM Trypsin - Dilute |
5 | 10 nM Trypsin - Dilute |
6 | 1 nM Trypsin - Dilute |
7 | Blank |
8 | 1 μM Trypsin |
9 | 100 nM Trypsin |
10 | 10 nM Trypsin |
11 | 1 nM Trypsin |
Gel BI
Well | Sample |
1 | Ladder |
2 | Blank |
3 | 1 μM Chymotrypsin - Dilute |
4 | 100 nM Chymotrypsin - Dilute |
5 | 10 nM Chymotrypsin - Dilute |
6 | 1 nM Chymotrypsin - Dilute |
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 - Dilute |
4 | 100 nM Thermolysin - Dilute |
5 | 10 nM Thermolysin - Dilute |
6 | 1 nM Thermolysin - Dilute |
7 | Blank |
8 | 1 μM Thermolysin |
9 | 100 nM Thermolysin |
10 | 10 nM Thermolysin |
11 | 1 nM Thermolysin |
- 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
- 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.
- The gel was run for approximately 40 min at 200 V, 0.05 A, and 10 W.
- After 40 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes
- The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 20 minutes
- Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) for 20 minutes and the results are pictured below:
Results
NOTE
- Only the B side gel appeared to be running for the first approx. 20 minutes; adjustments were made but the A side gels ultimately only ran for about 20 min instead of the suggested 40.
- The fixing, staining, and de-staining processes were shortened because of time limitations. Additionally, the results demonstrate what appears to be primarily protein ladder/marker that made its way into adjacent wells, and nothing else.