Difference between revisions of "User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/25"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
 
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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#*Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
 
#*Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
 
#Repeat last part of step 4 twice.
 
#Repeat last part of step 4 twice.
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Sample Set-up of the electrophoresis (SDS-PAGE)
 
Sample Set-up of the electrophoresis (SDS-PAGE)
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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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Latest revision as of 22:24, 26 September 2017

Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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