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 Co-IP & ELISA prep
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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
- 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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Co-IP & ELISA prep
