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Protocol
Effector caspase activation assay
Keywords: caspase activity
Assay for analysis of caspase3/7 specific activity
Required Materials
- Cells , to be analysed
- 24 well plates
- Cell culture media , in accordance with cell type
- Pefabloc
- Eppendorf tubes
- 96 well plates
- Synergy HT Multi-Mode Microplate Reader
- Rocking table
Main steps
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Media is removed from cells seeded on 24 well plates.
- (1VC07B7)
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Place tray on ice and leave on rocking table (20-30 rpm).
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Transfer 380 ul lysate to epp tubes (on ice).
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Transfer 40 ul lysate to black ½ area costar 96 well plates (on ice).…
- (1VBXNC6)
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Place the tray in a fluorometric kinetic plate reader (Such as Synergy…
Instructions
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Media is removed from cells seeded on 24 well plates.
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400 ul caspase lysis buffer (CLB) + additional 1:200 pefabloc is added / well
Recipie for caspase lysis buffer to use in Caspase activity assay.
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Mix well. Store at room temperature.
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Place tray on ice and leave on rocking table (20-30 rpm).
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Transfer 380 ul lysate to epp tubes (on ice).
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Transfer 40 ul lysate to black ½ area costar 96 well plates (on ice).
Avoid exposure to light!
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Add 40 ul caspase reaction mix (on ice).
Recipie for caspase reaction mix to use in Caspase activity assay.
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Mix well. Store dark, at room temperature.
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Place the tray in a fluorometric kinetic plate reader (Such as Synergy HT, Biotek) and initiate the caspase measurement program.
- Approximately readings every 20 sec over a 15 minutes interval.
- For data analysis ensure linear range of substrate cleavage.
