In Cell Western Protocol for Licor Odyssey

In cell Western protocol for Licor Odyssey: Volumes in 96-well should be typically 100-150 µl, but for expensive reagents (primary and secondary antibodies) 50 µl may be enough?

1. Wash 1x in PBS 2. Fix in 4 % paraformaldehyde in PBS for 20 min at 4 ˚ C 3. Permeabilize by 5 washes in 0.1 % Triton X-100 in PBS. Each wash is for 5 min on a shaker 4. Block non-specific with 5% BSA in PBS for 1.5 hrs (shaking) 5. Incubate in primary antibodies (pSer15 rabbit polyclonal 1:500 + -tubulin 1:1000 monoclonal) in PBS/ 5 % BSA (include control which has no primary antibody, only Odyssey Blocking buffer). Incubate 2 hrs with gentle shaking. 6. Wash 5 times with 0.1 % Tween-20 in PBS. Each wash is for 5 min. on a shaker 7. Dilute secondary antibodies in Odyssey Blocking buffer: Goat anti-rabbit: 1:5000, Goat anti-mouse 1:5000. Wrap in metal foil. Incubate 1 hr with shaking 8. Wash 5 times with 0.1 % Tween-20 in PBS. Each wash is for 5 min. on a shaker 9. After final wash remove all traces of wash solution. Turn plate upside down and tap. Scan plate immediately or store at 4 ˚ C.