I got a great explanation today on why it may be that my westerns haven't been detecting cleaved caspase-1. My conditions may also be contributing: positive control 1ug/ml LPS 5mM ATP, or treatment with bacteria for 0, 12, or 24 hours.
Any tips on detection of extracellular caspase-1 from THP-1s?
Eric,
In case you are still having problems detecting cleaved caspase1, here is my protocol. I plate my cells @ 1x10^6 in 12 well plates and infect or treat in 500ul medium. After treatment or infection, I remove 200ul of supernatant for ELISA and add 100ul 2xRIPA buffer and 125ul 4x SDS loading buffer to the cells+remaining supernatant(300ul). I boil the samples for 20min. They are then ready to load. I also use the anti-caspase-1 antibodies from adipogen. They work well.
If you are trying to immunoprecipitate caspase-1, the active form will mainly be in the culture supernatant. If you are trying to coimmunoprecipitate something with caspase-1, the best way I have found is to use a catalytically inactive mutant caspase-1 or to add YVAD or ZVAD inhibitors to the cells to prevent caspase-1 clevage. The complexes should still form, but will be more stable as once caspase-1 is active, the cells rapidly die and it is released into the culture medium. I hope this helps.
Hi Eric,
is your Ab designed to detected the cleaved forms and is your Ab specific for your antigen?
Eric,
In case you are still having problems detecting cleaved caspase1, here is my protocol. I plate my cells @ 1x10^6 in 12 well plates and infect or treat in 500ul medium. After treatment or infection, I remove 200ul of supernatant for ELISA and add 100ul 2xRIPA buffer and 125ul 4x SDS loading buffer to the cells+remaining supernatant(300ul). I boil the samples for 20min. They are then ready to load. I also use the anti-caspase-1 antibodies from adipogen. They work well.
If you are trying to immunoprecipitate caspase-1, the active form will mainly be in the culture supernatant. If you are trying to coimmunoprecipitate something with caspase-1, the best way I have found is to use a catalytically inactive mutant caspase-1 or to add YVAD or ZVAD inhibitors to the cells to prevent caspase-1 clevage. The complexes should still form, but will be more stable as once caspase-1 is active, the cells rapidly die and it is released into the culture medium. I hope this helps.
Yes. Any of the normal loading controls will work (Tubulin, actin, GAPDH).
- Badges
- Science method