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 Project name
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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 μL Leupeptin
- 1 μL Pepstatin (A)
- 5 μL Na3VO4
- 5 μL NaF
- PBS
- 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)
Method
- Use cells on S+ refreshed 12March2010
- 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)
Co-immunoprecipitation part I
- Beads were coated with anti-PKA antibodies.
- For 4 samples
- 480 μL beads
- Spin down and remove EtOH
- Wash with excess (400 μL) PBS
- Spin down and remove PBS
- Add 400 μL PBS
- Add 1.8 μL antibody (or not for control)
- Incubate with antibody ON @ 4 °C
S0 hTERT
- New batch of Ht31P to use
Coating of ELISA plate
- Mix
- 24 mL coating buffer
- 80 μL primary antibody
- Add 100 μL per well (96-wells plate)
- Incubate ON
96-wells plate ELISA
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04
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05
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10
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12
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| A
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| B
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| D
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| E
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| F
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| G
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| H
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Notes
Results
Conclusion
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Project name
