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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-IP & ELISA prep
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

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

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
 * BCA Protein Assay Reagent (bicinchoninic acid)
 * 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)

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

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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