Difference between revisions of "User:Moira M. Esson/Notebook/CHEM-571/2013/09/25"

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(Electrophoresis)
(Electrophoresis)
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==UV-vis==
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Figure 1. Corrected Absorbance Spectra of   
  
  

Revision as of 09:44, 25 September 2013

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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