User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/22

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cAMP precipitation Main project page
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Summary

Western blot

  • Prepare 6x 8% gels
  • Load 4 gels
Gel loading hTERT D9 + HEK293 see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec.
X HEK293
(20 μL)
Marker
(8 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)
S1
(20 μL)
S2
(20 μL)
S3
(20 μL)
X
  • 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
  • Upper 3 bands were not completely transferred to the membrane

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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