User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/23: Difference between revisions

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==Conclusion==
==Conclusion==
*{{done}} Ow..
*{{done}} Ow..
==Related==
===Related entries===
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|Co-IP part I & Western blot]]
===Same actions===
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24| Cell lysis & Co-IP Day II]]




Revision as of 01:12, 23 March 2010

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Summary

  • Cell lysis Co-IP
  • Co-IP Day II
  • Stimulation
    • ELISA
    • VASP
  • Western blot, secondary antibody
  • CHECK AVAILABILITY WESTERN BLOT THURSDAY!
  • REGISTER FOR MLII 24/3/10

Materials & Methods

Materials

Method

Determination of protein concentration

  • Use cells put on S0 23Feb2010
  • Put cells on ice
  • Remove medium
  • Wash twice with 5 mL cold PBS
  • Add 1 mL RIPA buffer
  • Scrape of cells and collect in 1.5 mL tube
  • Sonicate (4x short pulse)
  • Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)

Co-immunoprecipitation

  • resuspend beads prepared 23Feb2010
  • Prepare for all donors (D9 & D12)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

(possible also beads (30 μL) with only sample (200 μL))

  • Incubate ON @ °C
6-wells
# 1 2 3
A
B
24-wells
# 1 2 3 4 5 6
A
B
C
D
96-wells plate
# 01 02 03 04 05 06 07 08 09 10 11 12
A
B
C
D
E
F
G
H
I

Results

  • WHAT?

Conclusion

  • DONE Ow..

Related

Related entries

Same actions



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