User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/06: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">cAMP precipitation gel</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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* Proteins were transferred using the NuPage system (transfer buufer +10% methanol), 30 V for 1.5 h
* Proteins were transferred using the NuPage system (transfer buffer +10% methanol), 30 V for 1.5 h
** All of the markers was transferred to blot, to check gel was Coomassie stained after blotting
** All of the markers was transferred to blot, to check gel was Coomassie stained after blotting
* cAMP precipitation Ponceau S staining
* cAMP precipitation Ponceau S staining

Revision as of 05:30, 29 July 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

cAMP precipitation gel <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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

cAMP precipitation

  • Beads were washed as described 09April2010
  • 50 μL of NuPage LDS sample buffer + DTT was added to the beads (800 μL LDS sample buffer + 200 μL 1 M DTT)
    • Beads were put to 70 °C for 10 min.
  • Samples were run on NuPage prefabricated Bis-Tris gradient (4-12%) gel in MES buffer (with 500 μL Antioxidant in inner chamber (~200 mL)). 200 V.
Gel loading hTERT D9 + HEK293 & HeLa -S see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gel
cAMP prec. 		X X Invitrogen Marker
(8 μL) 		HeLa -S
(20 μL) 		HEK293
(20 μL) 		Biorad Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(20 μL) 		S2
(20 μL) 		S3
(20 μL) 		X X X
  • Proteins were transferred using the NuPage system (transfer buffer +10% methanol), 30 V for 1.5 h
    • All of the markers was transferred to blot, to check gel was Coomassie stained after blotting
  • cAMP precipitation Ponceau S staining


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