User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/31: Difference between revisions
From OpenWetWare
No edit summary |
No edit summary |
||
(4 intermediate revisions by the same user not shown) | |||
Line 42: | Line 42: | ||
| align="center" style="background:#f0f0f0;"|'''15''' | | align="center" style="background:#f0f0f0;"|'''15''' | ||
|- | |- | ||
| Sample||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter | | Sample||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter ]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||x||x||x||x||x||x||x | ||
|- | |- | ||
|} | |} | ||
Line 63: | Line 63: | ||
| align="center" style="background:#f0f0f0;"|'''15''' | | align="center" style="background:#f0f0f0;"|'''15''' | ||
|- | |- | ||
| Sample||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter | | Sample||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||Marker||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/08|hTERT RIPA]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/07|hTERT Potter]]||[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/27|HEK293 RIPA]]||x||x||x||x||x||x||x | ||
|- | |- | ||
|} | |} | ||
==== | ====Splitting cells==== | ||
* Splitting cells into 12-wells? | * <del>Splitting cells into 12-wells?</del> | ||
*D9 to passage 25 | |||
====<del>Immunoblot</del>==== | |||
*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 | |||
====<del>Preparing film</del>==== | |||
* 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) | |||
* Place blot in between plastic | |||
* Take picture (depends on what is available) | |||
<!--==Materials & Methods== | <!--==Materials & Methods== | ||
===Materials=== | ===Materials=== | ||
Line 79: | Line 91: | ||
--> | --> | ||
==Notes== | ==Notes== | ||
*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 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== | ||
*{{todo| WHAT?}} | *{{todo| WHAT?}} |
Revision as of 09:11, 31 May 2010
<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>
SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293) | <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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SummarySDS-PAGE
Splitting cells
|