Calpain Activity Assay Kit (ab65308)
Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Overview
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Product name
Calpain Activity Assay Kit -
Detection method
Fluorescent -
Sample type
Tissue Extracts, Cell Lysate -
Assay type
Quantitative -
Assay time
2h 00m -
Species reactivity
Reacts with: Mammals, Other species -
Product overview
Calpain Activity Assay Kit ab65308 provides optimized buffers and reagents for a convenient measurement of calpain activity.
The extraction buffer included in the kit specifically extracts cytosolic proteins without contamination by cell membrane and lysosome proteases. It also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain within the cytosol.
The calpain activity assay protocol is based on the detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.
Calpain activity assay protocol summary:
- add samples and positive and negative controls to wells
- add reaction buffer and calpain substrate
- incubate for 60 min
- analyze with a microplate reader -
Notes
If additional Ac-LLY-AFC substrate is needed, it can be purchased separately as ab171379.
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Platform
Microplate reader
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components Identifier 100 tests 10X Reaction Buffer Clear 1 x 1.5ml Active Calpain I (Positive Control) Green 1 x 10µl Calpain Inhibitor Z-LLY-FMK Orange 1 x 10µl Calpain Substrate Ac-LLY-AFC Amber 1 x 500µl Extraction Buffer WM 1 x 25ml -
Research areas
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Function
Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. -
Tissue specificity
Ubiquitous. -
Sequence similarities
Belongs to the peptidase C2 family.
Contains 1 calpain catalytic domain.
Contains 4 EF-hand domains. -
Cellular localization
Cytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding. - Information by UniProt
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Alternative names
- Ca2 activated neutral protease
- Calcium activated neutral proteinase
- Calcium activated neutral proteinase small subunit
see all
Images
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Functional assays: Calpain Activity Assay Kit (ab65308)
Different amounts of positive control (Calpain I) treated with 1 μL of inhibitor (Z-LLY-FMK), background signal subtracted, duplicates; +/- SD.
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Functional assays: Calpain Activity Assay Kit (ab65308) -
Functional Studies - Calpain Activity Assay Kit (ab65308)Typical Data for ab65308: Active Calpain (1 µg) was incubated at 37 °C for one hour using the Calpain Substrate with or without 20 µM Calpain Inhibitor.