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 cAMP precipitation
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Summary
SDS-PAGE
Washing beads: cAMP precipitation
- Spin beads 5 min. 2100xg @ 4 °C
- Collect supernatant (= NAC)
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump and syringe
- Add 50 μL of 4x sample buffer
- Spin 5 min. 2100xg @ 4 °C
RIPA BUFFER (100 mL)
- 50 mM Tris-HCl (5 mL 1 M Tris pH 7.4)
- Adjust to pH 7.4
- 150 mM NaCl (0.87 g NaCl)
- 1 mM EDTA (37.22 mg EDTA)
- 1% Triton x-100 (1 mL Tritox x-100)
- 1% Sodium deoxycholate (1,03 g (97%))
- 0.1% SDS (1 g SDS)
Notes
- One gel was too small, probably added water to fast (to remove airbubbles from top). I only needed 5 so it's okay.
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cAMP precipitation
