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 cAMP precipitation
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Summary
Western blot
- Prepare 6x 8% gels
- Load 4 gels
Gel loading hTERT D9 + HEK293 see codes 21June2010
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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Gel
cAMP prec.
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X
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HEK293
(20 μL)
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Marker
(8 μL)
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C1
(20 μL)
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C2
(20 μL)
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C3
(20 μL)
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S1
(20 μL)
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S2
(20 μL)
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S3
(20 μL)
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X
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- Transfer to blot
- Block blot ON in 1% BSA in TBS-T
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.
- Second wash of beads was kept in centrifuge for ~30 min. and centrifuged a second time before continuing to third wash
- Third wash was performed with new RIPA buffer
- Gel running was troublesome, first a bit blurry but straightened out later. Marker was a bit faint and running took >2 h.
- HiMark (Invitrogen) was used
Results
- Ponceau staining of cAMP precipitations 4 gels with the same samples and marker
Discussion
- Nothing visible, HOPEFULLY this is due to a low amount of protein (or bad Ponceau) and NOT due to the complete lack of protein
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cAMP precipitation
