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Potter-Elvehjem Cell Homogenizing
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Summary
- cAMP precipitation of D9 P28 treated with 15% CSE for RII overlay. New way of disrupting cells was used that would be more gently than the previous method.
Materials & Methods
Materials
- Potter buffer
- 250 mM Sucrose (8.56 g sucrose/sacharose in 100 mL)
- 3 mM imidazole (300 μL 1 M imidazole in 100 mL)
- Potter lysis buffer (per mL)
- 1 mL Potter buffer
- 8 μL protease inhibitor mix
- 1 μL NaF
- 1 μL NaVO3
- 12.5 μL PMSF
- 8-AHA-cAMP coated agarose beads
- Cold PBS
Method
Disrupting cells
- Put cells on ice
- Remove medium
- Wash twice with 6 mL cold PBS
- Add 0.5 mL Potter lysis buffer
- Scrape off cells and pool in 15 mL tube
- Using Potter-Elvehjem Homogenizer for 10-20 strokes @ 700 RPM gently homogenize cells
- Leave cells on ice @ 4 °C for 15 min.
- Centrifuge 15 min. @ 3000 x g and collect supernatant
Incubating lysates
- incubate 1.5 mL of each sample with 29.3 mg cAMP for 15-30 min. while rotating @ 4 °C
- Collect 100 mL of these samples
- Put 1.5 mL of each sample on 30 uL of 8-AHA-cAMP beads and incubate 2 – 4 h while rotating @ 4 °C
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
Samples on gel
- 75 μL lysate was mixed with 25 μL 4x sample buffer
- Samples were incubated @ 95 °C for 5 min.
- Samples were put to 8% gel together with remaining lysates on Sunday
Notes
- 50 μL of beads were used instead of 30 μL
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