- 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 was run on a Bio-Rad mini protean system with pre-cast TGX gels.
- Used Coomassive blue staining for the development
- 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
||0.5 hrs Pepstatin
||1 hr Pepsin
||1 hr Pepstatin
||1.5 hrs Pepstatin
Figure 1. Corrected Absorbance Spectra of Pepsin with Hemoglobin
Figure 2. Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin