User:Moira M. Esson/Notebook/CHEM-571/2013/09/25

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Objectives

  1. Run electrophoresis(SDS-PAGE) of pepsin and pepstatin samples prepared on 2013/09/24
  2. 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:

  1. 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
  2. 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
  3. Run electrophoresis for 30 minutes at 200V
  4. 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
  5. 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


UV-vis

Figure 1. Corrected Absorbance Spectra of Pepsin with Hemoglobin
Hemopepsin09251013zem.png

  • Note: For the 2hr sample some pieces of protein may have been present when running the sample.


Figure 2. Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
Pepstathemogl09252013zem.png

Notes

  • For both of the UV-vis spectra, there is no observable trend for the absorbance(the absorbance does not increase or decrease consistently with time). This may be due to the inclusion of protein in some of the sample runs. Another trial of these samples will be performed