User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/31: Difference between revisions
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====Splitting cells==== | ====Splitting cells==== | ||
* Splitting cells into 12-wells? | * <del>Splitting cells into 12-wells?</del> | ||
*D9 to passage 25 | *D9 to passage 25 | ||
====Immunoblot==== | ====<del>Immunoblot</del>==== | ||
*Block membrane in 1% BSA TBST solution for >1 h @ RT | *Block membrane in 1% BSA TBST solution for >1 h @ RT | ||
*Incubate with primary antibody 2 h @ RT or ON @ 4 °C | *Incubate with primary antibody 2 h @ RT or ON @ 4 °C | ||
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**1:10.000 dilution in blocking solution | **1:10.000 dilution in blocking solution | ||
*Wash 3x 10 min. with blocking solution | *Wash 3x 10 min. with blocking solution | ||
====Preparing film==== | ====<del>Preparing film</del>==== | ||
* 1:1 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions (2 mL per membrane) | * 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) | * Incubate membrane in ECL solution 5 min, RT (both sides of membrane) | ||
| Line 95: | Line 95: | ||
*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 | ||
*Gel #1 has the front of the loading buffer a bit blurry around 40 ~ 75 kDa | *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 | |||
==Results== | ==Results== | ||
Revision as of 09:11, 31 May 2010
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 SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293)
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SummarySDS-PAGE
Splitting cells
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