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)
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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>

Revision as of 13:34, 1 October 2013

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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




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