Crac Supernatant Preparation

- use a CRAC overexpressing cell line, grow to 5 x10⁶ cells/ml

- starve for 2 hours at 2 x 10⁷ cells/ml in DB

- from this point on, do everything on ice

- wash once in PM and resuspend at 8 x 10⁷ cells/ml in SLB (10 mM Tris pH 7.5; 0.2 mM EGTA; 200 mM sucrose)

- lyse using 5 mm nucleopore filters

- spin 9000 rpm (SS34) for 20 min

- store supernatant at -80°C

 

Notes:

- once in PM always keep on ice

- avoid bubbles

- absolutely no vortexing

- supernatants can be freeze/thawed (on ice) 3-4 times without significant loss of activity

 

This page was last edited 06/26/2003