User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/31: Difference between revisions

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====siRNA prep====
====Splitting cells====
* Splitting cells into 12-wells?
* 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 [[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 & Methods==
===Materials===
===Materials===
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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
*Wells of gel #1 were not great in that they appeared not to be perfectly separated at the bottom

Revision as of 03:54, 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

Results

  • WHAT?

Discussion

  • DONE Ow..


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