User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/25

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Co-immunoprecipitation part III Main project page
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Summary

The samples have been incubated with the (PKA) antibody coated beads and today the beads are washed and stored in Laemli buffer

Materials & Methods

Materials

  • Laemli buffer
  • PBS
  • syringe
  • Hot lysis buffer


Method

Co-immunoprecipitation

  • Centrifuge sample incubated with beads, 5 min. 14.000 RPM
  • Remove supernatant* (w. syringe)
  • Add 1 mL PBS
  • Centrifuge, 5 min. 14.000 RPM
  • Remove supernatant*
  • Add 75 μL Laemli buffer to beads
  • Store samples @ -20 °C
    • Alternatively go directly on to Western blot (Boil samples and spin beads down, bring supernatant onto gel)


* Be careful not to touch beads

VASP

  • Use cells grown by Bachelor students TODO
  • Remove medium
  • Wash with PBS twice and remove
  • Add 600 μL S0 or S0+Ht31 (see hTERT VASP schedule below)
  • Incubate 15-20 min. RT
  • Add 600 μL S0 or S0+6-Bnz (see hTERT VASP schedule below)
  • Incubate 10 min. RT
  • Add 600 μL S0 or S0 45% CSE (see hTERT VASP schedule below)
  • Incubate 10 min. RT
  • Remove medium
  • Wash with PBS and remove
  • Add 200 μL Hot Lysis buffer and scrape cells with tip
  • Transfer cell lysate into 1.5 mL tubes
  • Store @ -20 °C
hTERT VASP
#
1
2
3
A
Control
Ht31 (50 μM)
CSE (15%)
B
CSE (15%) + Ht31 (50 μM)
CSE (15%) + 6-Bnz (500 μM)
CSE (15%) + 6-Bnz (500 μM)+ Ht31 (50 μM)

Coating of plate

  • Prepare coating buffer
    • 1.24 g Na2CO3 in 100 mL (Buffer A)
    • 2.52 g NaHCO3 in 300 mL (Buffer B)
    • Take 70 mL of Buffer A
    • Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
  • Continue as described 18Feb2010

BSc.

  • Alamar blue
  • Stop stimulation (hTERT)
  • Cells to 96-wells (hTERT)

Results

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

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