User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/24

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Co-IP Part III, Stimulation stop/Viability staining Main project page
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Summary

  • Co-IP day III
  • Stop stimulation --> 12.41
  • Coat ELISA plate

Materials & Methods

Materials

  • Coating buffer
    • Solution A: 1.24 g Na2C03·H2O in 100 mL H2O
    • Solution B: 2.52 g NaHCO3 in 300 mL H2O
    • Take 70 mL of solution A
    • Add solution B until a pH of 9.6 is reached

Method

Co-immunoprecipitation day III

  • 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

Stop stimulation/Viability test

  • Collect 200 μL of supernatant for ELISA of each well
  • Remove the rest of the media
  • Wash twice with PBS
  • Add per well 475 μL HBSS + 25 μL Alamar blue
    • For ~50 wells that's 23.750 mL HBSS + 1250 μL Alamar blue
  • Incubate ~30 min. 37 °C
  • Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
  • This shows the metabolic (mitochondria) activity of cells

ELISA prep

Coating of plate
  • Mix twice
    • 24 mL coating buffer
    • 80 μL primary antibody
  • Add 100 μL per well (96-wells plate)
  • Incubate ON

Results

  • Viability staining
D9 P25 S0 S0 S0 HT31 HT31 HT31
CTR667667306896720873038327
CSE637353475520535555566517
CSE+8-p652652235092528747056222
CSE+Bnz732251494808496151475409

D12 P29 S0 S0 S0 HT31 HT31 HT31
CTR786275547341722062256267
CSE566747194365429043155527
CSE+8-p615351454494421239824774
CSE+Bnz60565707486341734351527

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