User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/31: 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 AKAP79 immunoblot & RII overlay (HEK293)</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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==Summary==
==Summary==
====SDS-PAGE====
====SDS-PAGE====
* HEK lysate, [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]] & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|Potter lysate]]
[[Image:Biorad_-_Precision_Plus_Protein_Dual_Color_marker.jpg|right|Biorad Precision Plus Protein Dual Color marker]]
* [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA lysate]], [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]] & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|Potter lysate]]
**HEK293 SDS-PAGE Sample was diluted with 4x loading buffer (25 μL buffer, 75 μL sample)
**All samples were denaturated @ 95 °C for 5 - 10 min.
* Put samples to 8% polyacrylamide gel
* Put samples to 8% polyacrylamide gel
* [http://www3.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@1364050109.1270810731@@@@&BV_EngineID=cccgadekdldelhkcfngcfkmdhkkdflm.0&categoryPath=%2fCatalogs%2fLife+Science+Research%2fElectrophoresis%2fProtein+Electrophoresis%2fElectrophoresis+Reagents+and+Standards%2fProtein+Standards+for+Electrophoresis%2fPrecision+Plus+Protein+Standards%2fPrecision+Plus+Protein+Prestained+Standards&catLevel=8&divName=Life+Science+Research&loggedIn=false&lang=English&country=IL&catOID=-26793&isPA=false&serviceLevel=Lit+Request Precision Plus Protein Dual Color marker]
*There was not enough sample to put everything on one gel, realized it at loading
** New samples were prepared for [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]] & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|Potter lysate]] (as described above for HEK293) with original lysates stored at -20 °C
* Antibodies to check AGAIN for AKAP79
** [http://www.scbt.com/datasheet-6445-akap-150-c-20-antibody.html AKAP 150 (C-20): sc-6445 goat polyclonal] (Santa Cruz, some overlap with AKAP79 epitope)
** [http://www.scbt.com/datasheet-17772-akap-79-d-9-antibody.html AKAP 79 (D-9): sc-17772 mouse monoclonal] (Santa Cruz)
**[http://www.millipore.com/coa/tech1/74jn74 AKAP79 rabbit monoclonal] (Upstate)
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''#'''
| align="center" style="background:#f0f0f0;"|'''#1'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''2'''
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| align="center" style="background:#f0f0f0;"|'''15'''
| align="center" style="background:#f0f0f0;"|'''15'''
|-
|-
| Sample||Marker||CSE beads||CSE NAC||CSE Lysate Potter||CSE Lysate RIPA||x||HEK293 RIPA lysate||marker||S<sup>0</sup> beads||S<sup>0</sup> NAC||S<sup>0</sup> Lysate Potter||S<sup>0</sup> Lysate RIPA||x||x||x
| Sample||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]]||x||x||x||x||x||x||x
|-
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''#2'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| 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'''
| align="center" style="background:#f0f0f0;"|'''13'''
| align="center" style="background:#f0f0f0;"|'''14'''
| align="center" style="background:#f0f0f0;"|'''15'''
|-
| Sample||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]]||x||x||x||x||x||x||x
|-
|-
|}
|}
====siRNA prep====
* Splitting cells into 12-wells?


==Materials & Methods==
====Splitting cells====
* <del>Splitting cells into 12-wells?</del>
*D9 to passage 25
====<del>Immunoblot</del>====
*Block membrane in 1% BSA TBST solution for >1 h @ RT
*Incubate with primary antibody 2 h @ RT or ON @ 4 °C
**1:1000 dilution of antibody in blocking solution
*Wash blot 10 min. with blocking solution
*Incubate with secondary antibody 1 h @ RT
** anti-rabbit, anti-mouse or anti-goat
**1:10.000 dilution in blocking solution
*Wash 3x 10 min. with blocking solution
====<del>Preparing film</del>====
* 1:1 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions (2 mL per membrane)
* Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
* Place blot in between plastic
* Take picture (depends on what is available)
<!--==Materials & Methods==
===Materials===
===Materials===
*  
*  


===Method===
===Method===
{| border="1"
 
|+'''6-wells'''
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==Notes==
==Notes==
*Wells of gel #1 were not great in that they appeared not to be perfectly separated at the bottom
*Gel #1 has the front of the loading buffer a bit blurry around 40 ~ 75 kDa
*Gel #1 took forever to run (3h and stil not completely done), and it was not the only one in the lab, so diffulties in SDS-PAGE not completely gone. Gel #2 cancelled for today
==Results==
==Results==
*{{todo| WHAT?}}
*{{todo| WHAT?}}
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*{{done}} Ow..
*{{done}} Ow..


==Related==
<!--==Related==
===Related entries===
===Related entries===
===Same actions===
===Same actions===
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__NOTOC__
__NOTOC__

Revision as of 09:11, 31 May 2010

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

SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293) <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

SDS-PAGE

Biorad Precision Plus Protein Dual Color marker
Biorad Precision Plus Protein Dual Color marker
#1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample Marker hTERT RIPA hTERT Potter HEK293 RIPA Marker hTERT RIPA hTERT Potter HEK293 RIPA x x x x x x x
#2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample Marker hTERT RIPA hTERT Potter HEK293 RIPA Marker hTERT RIPA hTERT Potter HEK293 RIPA x x x x x x x

Splitting cells

  • Splitting cells into 12-wells?
  • D9 to passage 25

Immunoblot

  • Block membrane in 1% BSA TBST solution for >1 h @ RT
  • Incubate with primary antibody 2 h @ RT or ON @ 4 °C
    • 1:1000 dilution of antibody in blocking solution
  • Wash blot 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-rabbit, anti-mouse or anti-goat
    • 1:10.000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution

Preparing film

  • 1:1 ratio ECL solutions (2 mL per membrane)
  • Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
  • Place blot in between plastic
  • Take picture (depends on what is available)

Notes

  • Wells of gel #1 were not great in that they appeared not to be perfectly separated at the bottom
  • Gel #1 has the front of the loading buffer a bit blurry around 40 ~ 75 kDa
  • Gel #1 took forever to run (3h and stil not completely done), and it was not the only one in the lab, so diffulties in SDS-PAGE not completely gone. Gel #2 cancelled for today

Results

  • WHAT?

Discussion

  • DONE Ow..


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