User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/01: Difference between revisions

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{|{{table}} width="800"
{|{{table}} width="800"
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|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;">SDS-PAGE for immunoblot (fail)</span>
|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>}}
|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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* Gel #3 was prepared
* Gel #3 was prepared
** All other gels stored at 4 °C were discarded
** All other gels stored at 4 °C were discarded
** To prevent more wasting of samples only marker and loading buffer (4x) was used
** seemed to run owkay after also running buffer was renewed
* Immunotreatment of blot #2 was performed as described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/31|yesterday]]


==Materials & Methods==
==Materials & Methods==
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===Method===
===Method===
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|+'''6-wells'''
| align="center" style="background:#f0f0f0;"|'''#2'''
!#
| align="center" style="background:#f0f0f0;"|'''1'''
!<u>1</u>
| align="center" style="background:#f0f0f0;"|'''2'''
!<u>2</u>
| align="center" style="background:#f0f0f0;"|'''3'''
!<u>3</u>
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#f0f0f0;"|'''6'''
| align="center" style="background:#f0f0f0;"|'''7'''
| align="center" style="background:#f0f0f0;"|'''8'''
| align="center" style="background:#f0f0f0;"|'''9'''
| align="center" style="background:#f0f0f0;"|'''10'''
| align="center" style="background:#f0f0f0;"|'''11'''
| align="center" style="background:#f0f0f0;"|'''12'''
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| align="center" style="background:#f0f0f0;"|'''14'''
| align="center" style="background:#f0f0f0;"|'''15'''
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!A
| Sample||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]
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==Notes==
==Notes==
==Results==
==Results==

Revision as of 13:14, 19 August 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

SDS-PAGE for immunoblot (fail) <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Gel #2 was tried with fresh APS 10% (previous was 1 month old)
    • Still large blur was seen during and after running the gel.
    • When checking pH of the stacking gel a pH of 7.15 was found instead of 6.8
    • This will prevent the stacking function and would explain the blur seen while running
  • New stacking buffer was prepared
    • 18.9 g TRIS
    • 150 mL dH2O
    • pH 6.8
    • added to 250 mL with dH2O
  • Gel #3 was prepared
    • All other gels stored at 4 °C were discarded
    • To prevent more wasting of samples only marker and loading buffer (4x) was used
    • seemed to run owkay after also running buffer was renewed
  • Immunotreatment of blot #2 was performed as described yesterday

Materials & Methods

Materials

Method

#2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample hTERT RIPA hTERT Potter HEK293 RIPA Marker hTERT RIPA hTERT Potter HEK293 RIPA Marker hTERT RIPA hTERT Potter HEK293 RIPA Marker hTERT RIPA hTERT Potter HEK293 RIPA


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