Objectives
- Run electrophoresis(SDS-PAGE) of pepsin and pepstatin samples prepared on 2013/09/24
- Finish running UV-Vis samples of pepsin and pepstatin samples prepared on 2013/09/24
Electrophoresis
- Electrophoresis was run on a Bio-Rad mini protean system with pre-cast TGX gels.
- Used Coomassive blue staining for the development
General Protocol:
- Prepare the gel and assemble the electrophoresis cell
- Refer here for exact details on how to assemble the unit.
- Remove comb and tape from the gel
- Rinse the walls with running buffer (30.0 Tris, 44.1g glycine, 10g SDS, H2O to 1L)
- Assemble cell
- Fill the inner and outer buffer chambers with running buffer
- Load samples prepared yesterday
- Note: Also loaded two hemeglobin samples for comparison and pepsin and pepstatin reactions left overnight(these were prepared in the same fashion as 2013/09/24. In total ran 12 samples.
- Note: Loaded entire 20μL samples
- Run electrophoresis for 30 minutes at 200V
- Develop stain of the gel
- Place gel in fixant solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes to stop the proteins from migrating further
- Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
- Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
- Repeat last part of step 4 twice.
Table 1. Sample set up of electrophoresis gel
| Well Numbers
|
Sample
|
| 1 |
Hemoglobin
|
| 2 |
Pepsin
|
| 3 |
Pepstatin
|
| 4 |
0.5hrs Pepsin
|
| 5 |
0.5 hrs Pepstatin
|
| 6 |
1 hr Pepsin
|
| 7 |
1 hr Pepstatin
|
| 8 |
1.5hrs Pepsin
|
| 9 |
1.5 hrs Pepstatin
|
| 10 |
2hrs Pepsin
|
| 11 |
2hrs Pepstatin
|
| 12 |
Hemoglobin
|
|
|
|
Project name
