Caspase-1 Western spontaneous activation - Need help (Jan/24/2008 )
I am doing Western Blotting on HeLa cells to detect caspase-1. The antibody I'm using (Upstate cat#06-503) detects both the inactive form 45KD and the active form 20KD. Other papers have used this antibody to show caspase-1 activation via Western blotting in HeLa cells. I've been trying to use it, but every time I get caspase-1 activation (a 20KD band) in the control. I'm using Nigericin 20uM for 1h as a positive control but I'm not seeing any difference since the control already has caspase-1 activated. I tried changing media, changing antibody batch, cell confluency, protease cocktail, but nothing worked. Any ideas? Have this happened to anybody before. I really appreciate your input.
you have to keep in mind that some cells in all cultures are undergoing apoptosis (background). so its normal to get a cleaved casp3,7,8,9 or even 1 in control. you have to treat your cells for a longer period to see some more significant cleaved products than the control.
give it some time, say 3hrs minimum OR do a time course...for caspase studies time course experiments reveal many interesting mechanisms