User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/27

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] cAMP precipitation & AKAP79 immunodetection
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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)
 * 1) Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
 * 2) Add DTT just before adding membrane
 * 3) Completely immerse membrane
 * 4) Incubate 30 min @ 70 °C
 * 5) Wash 3x with dH2O or TBS-T
 * 6) 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

Results

 * AKAP79 immunoblot



Discussion

 * ✅ Ow..


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