User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/15

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Co-IP & ELISA prep Main project page
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Summary

  • Co-IP
    • Cell lysation
    • Preparation of beads
  • S0 24-wells
  • Coat ELISA Plate


Materials & Methods

Materials

  • RIPA buffer (per mL)
    • 1.06 mg β-glycerolphosphate
    • 1 mL RIPA buffer
    • 1 μL Apoprotein (1 mg/mL)
    • 1 μL Leupeptin (1 mg/mL)
    • 1 μL Pepstatin (A) (1 mg/mL)
    • 5 μL Na3VO4
    • 5 μL NaF (200 mM)
  • PBS
  • DMEM S0
  • Coomassie (Bradford) Protein Assay Kit
  • ELISA 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)
  • IL-8 coating antibody
  • 96-wells plate (MaxiSorb)

Method

Putting cells to S0

  • Wash cells twice with PBS
  • Add 500 μL DMEM S0 to each well
  • Incubate @ 37 °C

Determination of protein concentration

  • Use cells on S+ refreshed 12March2010
    • 1 Ø 10 cm dish each donor
  • 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)
  • Store samples @ -20 °C

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 2 samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
    • 280 μL beads
    • Spin down and remove EtOH
    • Wash with excess (400 μL) PBS
    • Spin down and remove PBS
    • Add 1:1 PBS:Beads (280 μL)
      • 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
      • 80 μL for Only Beads (D9/D12)
      • (30 μL for each condition)
    • Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL
    • Incubate ON @ 4 °C

Coating of ELISA plate

  • Mix
    • 24 mL coating buffer (date of preparation 11March2010)
    • 80 μL primary antibody
  • Add 100 μL per well (96-wells plate)
  • Incubate ON

Notes

  • 6-wells plates which had their medium refreshed 12March2010 were thrown out
  • In putting cells to S0 D12 well A2 got 1 mL instead of 0.5 mL DMEM S0, no large complications are to be expected.
  • Cells used for Co-IP and those put to S0 were
  • For the Pierce determination new vials B and C of the standard curve were prepared.

Results

  • Pierce determination Co-IP
    • D9: 2.43 mg/mL
    • D12: 2.10 mg/mL

Conclusion

  • There is enough protein present for Co-IP (>1mg/mL)



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