Dominik Haudenschild May 28, 2008 from JBC281(45):34716-24
1. Release cells from Alginate with 50mM EDTA in PBS for minimal time
2. Centrifuge cells 2 minutes 1000g to pellet
3. Wash cells once with HBSS to get rid of alginate and remaining EDTA
4. Incubate 30 minutes at 37 degrees in gentile lysis buffer
5. Centrifuge 2100g for 30 minutes to pellet insoluble protein fraction
6. Assay Supernatant (soluble) and Pellet (insoluble) by western blotting for Vimentin and protein-of-interest that might be vimentin-associated
a. Keep track of relative volumes loaded onto SDS-PAGE, so that an approximate calculation can be made for the percentage of Vimentin in the pellet versus supernatant
Gentile lysis buffer:
150mM NaCl
1% NP-40
10% glycerol
20mM HEPES pH 7.6
2mM Sodium Pyrophosphate
*just before use add
1:100 of Phosphatase Inhibitor 2 (Sigma P5726)
1:1000 of Protease Inhibitor (Sigma P-8340)
1mM PMSF