User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/08

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


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Summary

 * Short summary

Materials

 * 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
 * cAMP coated agarose beads

cAMP precipitation & Preparations RII overlay

 * Cells were lysed as described 24Feb2010 without determining protein concentration
 * 900 μL was put to cAMP coated agarose beads and incubated @ 4 °C ON while mixing
 * 75 μL was mixed with 25 μL 4x sample buffer and incubated @ 95 °C for 5 min.
 * Samples were put to -20 °C together with remaining lysate

Taking cells from -80 °C part II

 * D9 P21 had media removed and was washed twice with warm PBS
 * 10 mL warm DMEM S+ was added
 * D12 P26 was left to grow without disturbance due to lack of attachment

Related topics

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