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

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


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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)

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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