User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/07

 .todo { color: red } .done { color: green} {| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Potter-Elvehjem Cell Homogenizing
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Summary

 * cAMP precipitation of D9 P28 treated with 15% CSE for RII overlay. New way of disrupting cells was used that would be more gently than the previous method.

Materials

 * Potter buffer
 * 250 mM Sucrose (8.56 g sucrose/sacharose in 100 mL)
 * 3 mM imidazole (300 μL 1 M imidazole in 100 mL)
 * Potter lysis buffer (per mL)
 * 1 mL Potter buffer
 * 8 μL protease inhibitor mix
 * 1 μL NaF
 * 1 μL NaVO3
 * 12.5 μL PMSF
 * 8-AHA-cAMP coated agarose beads
 * Cold PBS

Disrupting cells

 * 1) Put cells on ice
 * 2) Remove medium
 * 3) Wash twice with 6 mL cold PBS
 * 4) Add 0.5 mL Potter lysis buffer
 * 5) Scrape off cells and pool in 15 mL tube
 * 6) Using Potter-Elvehjem Homogenizer for 10-20 strokes @ 700 RPM gently homogenize cells
 * 7) Leave cells on ice @ 4 °C for 15 min.
 * 8) Centrifuge 15 min. @ 3000 x g and collect supernatant

Incubating lysates

 * 1) incubate 1.5 mL of each sample with 29.3 mg cAMP for 15-30 min. while rotating @ 4 °C
 * 2) Collect 100 μL of these samples
 * 3) Put 1.5 mL of each sample on 30 uL of 8-AHA-cAMP beads and incubate 2 – 4 h while rotating @ 4 °C

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

Samples on gel

 * 1) 75 μL lysate was mixed with 25 μL 4x sample buffer
 * 2) Samples were incubated @ 95 °C for 5 min.
 * 3) Samples were put to 8% gel together with remaining lysates on Sunday