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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293)
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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SDS-PAGE

 * HEK293 RIPA lysate, hTERT RIPA & 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
 * 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 hTERT RIPA & Potter lysate (as described above for HEK293) with original lysates stored at -20 °C
 * Antibodies to check AGAIN for AKAP79
 * AKAP 150 (C-20): sc-6445 goat polyclonal (Santa Cruz, some overlap with AKAP79 epitope)
 * AKAP 79 (D-9): sc-17772 mouse monoclonal (Santa Cruz)
 * AKAP79 rabbit monoclonal (Upstate)

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)

Discussion

 * ✅ Ow..


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