User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/25: Difference between revisions
From OpenWetWare
(Autocreate 2013/09/25 Entry for User:Eleni_N._Kalivas/Notebook/CHEM-571) |
(fix raw html notebook nav) |
||
(3 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==Objective== | ||
* | *To run electrophoresis(SDS-PAGE) and UV vis on the pepsin and pepstatin prepared on 09/24/13. | ||
==Protocol== | |||
'''General Protocol''': | |||
# Prepare the gel and assemble the electrophoresis cell | |||
#*Refer [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf 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, H<sub>2</sub>O to 1L) | |||
#*Assemble cell | |||
#*Fill the inner and outer buffer chambers with running buffer | |||
#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 [[User:Moira_M._Esson/Notebook/CHEM-571/2013/09/24|2013/09/24]]. In total ran 12 samples. | |||
#*Note: Loaded entire 20μL samples | |||
#Run electrophoresis for 30 minutes at 200V | |||
#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 | |||
#Repeat last part of step 4 twice. | |||
Sample Set-up of the electrophoresis (SDS-PAGE) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Well Numbers''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|- | |||
| | |||
|} | |||
<br> | |||
Image of SDS Electrophoresis | |||
[[Image:FLUBBER.jpg|750px]] | |||
==UV vis== | |||
Corrected Absorbance Spectra of Pepsin with Hemoglobin | |||
[[Image:Hemopepsin09251013zem.png|750px]] | |||
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 | |||
[[Image:Pepstathemogl09252013zem.png|750px]] | |||
Latest revision as of 23:24, 26 September 2017
Project name | Main project page Previous entry Next entry | |||||||||||||||||||||||||||
Objective
ProtocolGeneral Protocol:
UV visCorrected 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
|