Cell culture supernatants were precipitated by adding an equal volume of methanol and 0.25 volumes of chloroform, vortexed and centrifuged at 20.000 × g for 10 min. The upper phase was discarded and 500 µl of methanol was added to the interphase. This mixture was centrifuged at 20.000 × g for 10 min and the protein pellet dried at 55 °C, resuspended in Laemmli buffer and boiled at 99°C for 5 min. Samples were separated by SDS-PAGE (15%) and transferred onto nitrocellulose membranes. As indicated, blots were incubated with rabbit polyclonal antibody to anti murine caspase-1 p10 (sc-514, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti human caspase-1 p10 (sc-515, Santa Cruz Biotechnology), rabbit polyclonal anti human cleaved IL-1β (Asp116) (Cell Signaling, Boston, MA) or rabbit polyclonal anti murine cathepsin B (R&D Systems, Minneapolis, MN).
Protocol detail
Caspase-1 and IL-1β Activation Assay
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Antibody
Caspase 1
Cathepsin b
Cell culture
Chloroform
Human
Il 1β
Interphase
Laemmli buffer
Membranes
Methanol
Murine
Nitrocellulose
Protein
Rabbit
Sc 514
Sds page
Corresponding Organization :
Other organizations : University of Massachusetts Chan Medical School, Ludwig-Maximilians-Universität München, Norwegian University of Science and Technology
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Variable analysis
independent variables
- Cell culture supernatants were precipitated by adding an equal volume of methanol and 0.25 volumes of chloroform, vortexed and centrifuged at 20.000 × g for 10 min.
- The upper phase was discarded and 500 µl of methanol was added to the interphase. This mixture was centrifuged at 20.000 × g for 10 min and the protein pellet dried at 55 °C, resuspended in Laemmli buffer and boiled at 99°C for 5 min.
- Samples were separated by SDS-PAGE (15%) and transferred onto nitrocellulose membranes.
- Rabbit polyclonal antibody to anti murine caspase-1 p10 (sc-514, Santa Cruz Biotechnology, Santa Cruz, CA)
- Rabbit polyclonal anti human caspase-1 p10 (sc-515, Santa Cruz Biotechnology)
- Rabbit polyclonal anti human cleaved IL-1β (Asp116) (Cell Signaling, Boston, MA)
- Rabbit polyclonal anti murine cathepsin B (R&D Systems, Minneapolis, MN)
- Independent variables not explicitly mentioned.
- Dependent variables not explicitly mentioned.
- Control variables not explicitly mentioned.
- Positive controls: Not specified.
- Negative controls: Not specified.
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