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 cAMP precipitation & AKAP79 immunodetection
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Summary
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
Immunoblot
- Wash blot incubated ON 3x 10 min. with blocking solution
- Incubate with secondary antibody 1 h @ RT
- anti-mouse
- 1:10.000 dilution in blocking solution
- Wash 3x 10 min. with blocking solution
Preparing film
- 1:1 ratio ECL solutions (2 mL per membrane)
- Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
- Place blot in between plastic
- Take picture (depends on what is available)
Stripping blot
- Stripping solution
- 50 mL dH2O
- 5 mL Tris-Cl (1.0 M, pH 6.8) (Stacking gel buffer SDS-PAGE)
- 1 g SDS
- 150 mg DTT (Add later)
- Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
- Add DTT just before adding membrane
- Completely immerse membrane
- Incubate 30 min @ 70 °C
- Wash 3x with dH2O or TBS-T
- Membrane can now be blocked again
Immunoblot
- Block membrane in 1% BSA TBST solution for >1 h @ RT
- Incubate with primary antibody ON @ 4 °C
- Antibodies used on membrane
- Rabbit monoclonal antibody - AKAP79 (Upstate)
- 1:1000 dilution in blocking solution
Lysing HEK293 cells
- RIPA (Lysis) Buffer (per 1 mL)
- 1.06 mg β-Glycerolphosphate
- 1 mL RIPA buffer
- 8 μL Protease inhibitor mix
- 1 μL NaF (1 M)
- 10 μL NaVO3 (10 mM)
- 12.5 μL PMSF
- Cells were lysed as described 24Feb2010 without determining protein concentration
Materials & Methods
Materials
Method
Notes
- HEK293 cells might have been a nice control to take along with the AKAP79 blot
- Antibody check would have been nice to compare to the old antibody
Results
Discussion
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cAMP precipitation & AKAP79 immunodetection
