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

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(Autocreate 2013/09/25 Entry for User:Eleni_N._Kalivas/Notebook/CHEM-571)
Current revision (13:30, 9 October 2013) (view source)
(Protocol)
 
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==Entry title==
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==Objective==
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* Insert content here...
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*To run electrophoresis(SDS-PAGE) and UV vis on the pepsin and pepstatin prepared on 09/24/13.
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==Protocol==
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'''General Protocol''':
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# Prepare the gel and assemble the electrophoresis cell
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#*Refer [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf here] for exact details on how to assemble the unit.
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#*Remove comb and tape from the gel
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#*Rinse the walls with running buffer (30.0 Tris, 44.1g glycine, 10g SDS, H<sub>2</sub>O to 1L)
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#*Assemble cell
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#*Fill the inner and outer buffer chambers with running buffer
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#Load samples prepared yesterday
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#*Note: Also loaded two hemeglobin samples for comparison and pepsin and pepstatin reactions left overnight(these were prepared in the same fashion as [[User:Moira_M._Esson/Notebook/CHEM-571/2013/09/24|2013/09/24]]. In total ran 12 samples.
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#*Note: Loaded entire 20μL samples
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#Run electrophoresis for 30 minutes at 200V
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#Develop stain of the gel
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#*Place gel in fixant solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes to stop the proteins from migrating further
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#*Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
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#*Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
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#Repeat last part of step 4 twice.
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Sample Set-up of the electrophoresis (SDS-PAGE)
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Well Numbers'''
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| align="center" style="background:#f0f0f0;"|'''Sample'''
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|-
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| 1||Hemoglobin
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|-
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| 2||Pepsin
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|-
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| 3||Pepstatin
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|-
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| 4||0.5hrs Pepsin
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|-
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| 5||0.5 hrs Pepstatin
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|-
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| 6||1 hr Pepsin
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|-
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| 7||1 hr Pepstatin
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|-
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| 8||1.5hrs Pepsin
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|-
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| 9||1.5 hrs Pepstatin
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|-
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| 10||2hrs Pepsin
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|-
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| 11||2hrs Pepstatin
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|-
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| 12||Hemoglobin
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|-
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|
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|}
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<br>
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Image of SDS Electrophoresis
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[[Image:FLUBBER.jpg|750px]]
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==UV vis==
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Corrected Absorbance Spectra of Pepsin with Hemoglobin
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[[Image:Hemopepsin09251013zem.png|750px]]
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NOTES: For the 2 hour sample some pieces of protein may have been present when running the sample.
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Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
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[[Image:Pepstathemogl09252013zem.png|750px]]
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 +
 

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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
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


Image of SDS Electrophoresis

UV vis

Corrected Absorbance Spectra of Pepsin with Hemoglobin 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




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