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

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(Electrophoresis)
Current revision (19:34, 7 October 2013) (view source)
(UV-vis)
 
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Figure 1. SDS-PAGE Gel
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[[Image:FLUBBER.jpg]]
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==UV-vis==
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Figure 2. Corrected Absorbance Spectra of Pepsin with Hemoglobin
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[[Image:Hemopepsin09251013zem.png]]
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*Note: For the 2hr sample some pieces of protein may have been present when running the sample.
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Figure 3. Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
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[[Image:Pepstathemogl09252013zem.png]]
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==Notes==
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*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
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*Placed the gel in 10mLs of fixing solutions. Actually ended up dying the gel for 1hour and 15mins.
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Current revision

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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
1Hemoglobin
2Pepsin
3Pepstatin
40.5hrs Pepsin
50.5 hrs Pepstatin
61 hr Pepsin
71 hr Pepstatin
81.5hrs Pepsin
91.5 hrs Pepstatin
102hrs Pepsin
112hrs Pepstatin
12Hemoglobin


Figure 1. SDS-PAGE Gel
Image:FLUBBER.jpg

UV-vis

Figure 2. Corrected Absorbance Spectra of Pepsin with Hemoglobin
Image:Hemopepsin09251013zem.png

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


Figure 3. Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
Image: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
  • Placed the gel in 10mLs of fixing solutions. Actually ended up dying the gel for 1hour and 15mins.



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