Note on detection of Caspase 12 activated fragment in ER stressed cells
Both the precursor and cleaved fragment of Caspase 12 can usually be clearly detetced by immunoblot when cells are treated for ~25-30hrs with tunicamycin, but this does require lysis of the cells directly in 2% SDS sample buffer (and not in the "typical" caspase lysis buffer of CHAPS and low pH).
We find that the best way to lyse the cells is to wash the plates twice in PBS then to tilt the plate sideways for 1 minute followed by aspiration of the remaining buffer. Then 100-250ul (for a 6 cm dish) of hot (95C) lysis buffer is added and swirled around the dish, which is then rapidly scraped. The lysate is transferred into a 1.5ml tube, boiled 5 minutes, and sheared with a 27-29 gauge needle or sonicated to break up the DNA.
Heather P. Harding
March 19, 2004