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

Summary

Western blot

• Prepare 6x 8% gels
• Load 4 gels

• 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