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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Stripping blots, qPCR AKAP8, refresh medium new cells, 2nd AB
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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
   [[Media:20082010_qPCR_AKAP8,_GAPDH.xls|Results qPCR AKAP8]]


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