User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/25: 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;">Coomassie staining</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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==Summary==
==Summary==
===COOMASSIE STAINING===
===Coomassie staining===
#Run SDS-PAGE (8%)
#Fix in fixing solution (40% EtOH, 10% Acetic Acid) 10 min. @ RT
#Fix in fixing solution (40% EtOH, 10% Acetic Acid) 10 min. @ RT
#Stain with staining solution () for 30 min. @ RT (Heat in Microwave to speed up process)
#Stain with staining solution (0.025% [[wikipedia:Coomassie|Coomassie]] (G-250), 10% acetic acid) for 30 min. @ RT (Heat in Microwave to speed up process)
#Destain in destaining solution () for as long a necessary
#Destain in destaining solution (10% acetic acid) for as long a necessary
##Put Absorbing material on top of gel to catch excess coomassie
##Put Absorbing material on top of gel to catch excess coomassie
 
<br>
{| border="1"
|+'''Gel loading hTERT D9 + HEK293 see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''
! align="center" style="background:#f0f0f0;"|#
! align="center" style="background:#f0f0f0;"|<u>1</u>
! align="center" style="background:#f0f0f0;"|<u>2</u>
! align="center" style="background:#f0f0f0;"|<u>3</u>
! align="center" style="background:#f0f0f0;"|<u>4</u>
! align="center" style="background:#f0f0f0;"|<u>5</u>
! align="center" style="background:#f0f0f0;"|<u>6</u>
! align="center" style="background:#f0f0f0;"|<u>7</u>
! align="center" style="background:#f0f0f0;"|<u>8</u>
! align="center" style="background:#f0f0f0;"|<u>9</u>
! align="center" style="background:#f0f0f0;"|<u>10</u>
|-
!Gel<br>cAMP prec.
| align="center"|[[Media:Biorad_-_Precision_Plus_Protein_Dual_Color_marker.jpg|Biorad Marker]]<br> (8 μL)
| align="center"|HEK293<br>(20 μL)
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Invitrogen Marker]]<br> (8 μL)
| align="center"|C1<br>(20 μL)
| align="center"|C2<br>(20 μL)
| align="center"|C3<br>(20 μL)   
| align="center"|S1<br>(20 μL)
| align="center"|S2<br>(20 μL)
| align="center"|S3<br>(20 μL)
| align="center"|X
|-
|}
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==Materials & Methods==
==Materials & Methods==
===Materials===
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==Notes==
==Notes==
* Sample C1 was loaded a little less then 20 μL probably with some of the beads in there because of a lack of sample volume
==Results==
==Results==
*{{todo| WHAT?}}
*The amount of protein in the samples apears to be very low, bands around the size of the RII subunits (50 ~ 55 kDa) can be barely seen. Also between 116 - 150 kDa a faint band is seen.
*500 kDa band appears to reside in the stacking gel still, next time run gel further (although I usually don't make such large stacking gels)
[[Image:25062010_cAMP_precipitation_Coomassie.png|500px]]
*For better resolution gel will be destained over the weekend


==Discussion==
==Discussion==
*{{done}} Ow..
*It was mentioned that the Perkin-Elmer device used for the Bradford method for protein concentration is somehow defect and not giving proper measurements. This might have led to the wrong protein concentrations and a too high dilution of the samples.
*Protein concentration needs to be re-determined
*Perhaps the bands seen ~116 kDa are Epac2, also due to the fact that they are downregulated after CSE induction


==Related==
==Related==
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===Related entries===
===Related entries===
===Same actions===
===Same actions===
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===Related topics===
===Related topics===
*[[Silver:_Coomassie_Stain]]
*[[Jacobs:Protocol_Protein_Gel_Staining_Using_Coomassie_Blue_Protein_Stain]]
*[[Sosnick:Rapid_Coomassie_Staining]]
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__NOTOC__
__NOTOC__

Revision as of 08:25, 25 June 2010

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

Coomassie staining <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

Coomassie staining

  1. Run SDS-PAGE (8%)
  2. Fix in fixing solution (40% EtOH, 10% Acetic Acid) 10 min. @ RT
  3. Stain with staining solution (0.025% Coomassie (G-250), 10% acetic acid) for 30 min. @ RT (Heat in Microwave to speed up process)
  4. Destain in destaining solution (10% acetic acid) for as long a necessary
    1. Put Absorbing material on top of gel to catch excess coomassie


Gel loading hTERT D9 + HEK293 see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec. 		Biorad Marker
(8 μL) 		HEK293
(20 μL) 		Invitrogen Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(20 μL) 		S2
(20 μL) 		S3
(20 μL) 		X

Notes

  • Sample C1 was loaded a little less then 20 μL probably with some of the beads in there because of a lack of sample volume

Results

  • The amount of protein in the samples apears to be very low, bands around the size of the RII subunits (50 ~ 55 kDa) can be barely seen. Also between 116 - 150 kDa a faint band is seen.
  • 500 kDa band appears to reside in the stacking gel still, next time run gel further (although I usually don't make such large stacking gels)

  • For better resolution gel will be destained over the weekend

Discussion

  • It was mentioned that the Perkin-Elmer device used for the Bradford method for protein concentration is somehow defect and not giving proper measurements. This might have led to the wrong protein concentrations and a too high dilution of the samples.
  • Protein concentration needs to be re-determined
  • Perhaps the bands seen ~116 kDa are Epac2, also due to the fact that they are downregulated after CSE induction

Related

Related topics


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