User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/23: Difference between revisions

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==Summary==
==Summary==
Cells were put to S<sup>0</sup> as was supposed to be done [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/22| yesterday]]. [http://en.wikipedia.org/wiki/Immunoprecipitation Co-immunoprecipitation] was performed
Cells were put to S<sup>0</sup> as was supposed to be done [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/22| yesterday]]. Beads for [http://en.wikipedia.org/wiki/Immunoprecipitation Co-immunoprecipitation] were prepared.
==Materials & Methods==
==Materials & Methods==
===Materials===
===Materials===
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** 3 mL of suspension was diluted in 42 mL of DMEM S<sup>+</sup>
** 3 mL of suspension was diluted in 42 mL of DMEM S<sup>+</sup>
** Cells were plated out 15 mL per flask
** Cells were plated out 15 mL per flask
====Co-immunoprecipitation====
====Co-immunoprecipitation part I====
*
* Beads were coated with anti-PKA antibodies.
{| border="1"
* For <b>4</b> samples
|+'''6-wells'''
** 480 μL beads
!
** Spin down and remove EtOH
!<u>1</u>
** Wash with excess (400 μL) PBS
!<u>2</u>
** Spin down and remove PBS
!<u>3</u>
** Add 400 μL PBS
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** Add 1.8 μL antibody (or not for control)
!A
** Incubate with antibody ON @ 4 °C
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===Related entries===
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====Run 2: Ht31 Dose/Effect Curve & S<sup>0</sup> D9/D12====
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*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19|Counting cells 19Feb2010]]
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===Same actions===
!B
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15| Putting cells to S<sup>0</sup>]]
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|+'''24-wells'''
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==Results==
*{{todo| WHAT?}}
 
==Conclusion==
*{{done}} Ow..


==Related topics==
==Related topics==

Revision as of 14:23, 18 March 2010

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

Putting cells to S0, Co-immunoprecipitation <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Summary

Cells were put to S0 as was supposed to be done yesterday. Beads for Co-immunoprecipitation were prepared.

Materials & Methods

Materials

  • DMEM S0
  • DMEM S+
  • Cells prepared 19Feb2010
  • PBS
  • HT4 cells (neuroblastoma cell line)

Method

Putting cells to S0

  • Cells were put to S0 as described 15Feb2010
    • Cells were washed three times with PBS

Splitting HT4 cells

  • Split HT4 cells as described for hTERT cells
    • Cells of three flasks were resuspended in total volume of 18 mL (3 times 1 mL Trypsin + 15 mL DMEM S+)
    • 3 mL of suspension was diluted in 42 mL of DMEM S+
    • Cells were plated out 15 mL per flask

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 4 samples
    • 480 μL beads
    • Spin down and remove EtOH
    • Wash with excess (400 μL) PBS
    • Spin down and remove PBS
    • Add 400 μL PBS
    • Add 1.8 μL antibody (or not for control)
    • Incubate with antibody ON @ 4 °C

Related entries

Run 2: Ht31 Dose/Effect Curve & S0 D9/D12

Same actions

Related topics



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