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

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

Summary

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)

#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

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

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