User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/20: Difference between revisions

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* qPCR AKAP8
* qPCR AKAP8
** 25 μL
** 25 μL
* Western blot
** Primary antibodies were put on blot #1 made [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/16|16August2010]] and used for AKAP450 and AKAP95 blot [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/08/19|18August2010]]
***Gravin 1:5000 (Rabbit, antibody ZDE10030 animal 122 antibody (Eurogentech antibody service))
***AKAP79 1:1000 (Rabbit, Upstate)
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==Materials & Methods==
==Materials & Methods==
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==Notes==
==Notes==
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==Results==
==Results==
*qPCR AKAP8
*qPCR AKAP8

Revision as of 12:14, 20 August 2010

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Stripping blots, qPCR AKAP8, refresh medium new cells, 2nd AB <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

  • Stripping solution
    • 50 mL dH2O
    • 5 mL Tris-Cl (1.0 M, pH 6.8) (Stacking gel buffer SDS-PAGE)
    • 1 g SDS
    • 150 mg DTT (Add later)
  1. Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
  2. Add DTT just before adding membrane
  3. Completely immerse membrane
  4. Incubate 30 min @ 70 °C
  5. Wash 3x with dH2O or TBS-T
  6. Membrane can now be blocked again
  • qPCR AKAP8
    • 25 μL
  • Western blot
    • Primary antibodies were put on blot #1 made 16August2010 and used for AKAP450 and AKAP95 blot 18August2010
      • Gravin 1:5000 (Rabbit, antibody ZDE10030 animal 122 antibody (Eurogentech antibody service))
      • AKAP79 1:1000 (Rabbit, Upstate)

Results

  • qPCR AKAP8

  • Western blot AKAP95, AKAP450, RIIα

Attachment

<html> <a href="http://openwetware.org/wiki/Image:20082010_qPCR_AKAP8,_GAPDH.xls"> 

  <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50">

</a> </html> Results qPCR AKAP8

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