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Revision as of 13:38, 1 October 2013
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- To run electrophoresis(SDS-PAGE) and UV vis on the pepsin and pepstatin prepared on 09/24/13.
- 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.
Sample Set-up of the electrophoresis (SDS-PAGE)
| 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
Corrected Absorbance Spectra of Pepsin with Hemoglobin
NOTES: For the 2 hour sample some pieces of protein may have been present when running the sample.
Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin