User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/25

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Objective

  • To run electrophoresis(SDS-PAGE) and UV vis on the pepsin and pepstatin prepared on 09/24/13.

Protocol

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.


Sample Set-up of the electrophoresis (SDS-PAGE)

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


Image of SDS Electrophoresis FLUBBER.jpg

UV vis

Corrected Absorbance Spectra of Pepsin with Hemoglobin Hemopepsin09251013zem.png 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 Pepstathemogl09252013zem.png