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
This product is manufactured by BioVision, an Abcam company and was previously called K240 Calpain Activity Fluorometric Assay Kit. K240-100 is the same size as the 100 test size of ab65308.
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 100 tests 10X Reaction Buffer 1 x 1.5ml Calpain I Positive Control 1 x 10µl Calpain Inhibitor 1 x 10µl Calpain Substrate 1 x 500µl Extraction Buffer III 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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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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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.
Datasheets and documents
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SDS download
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Datasheet download
References (74)
ab65308 has been referenced in 74 publications.
- Chen X et al. Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity. Neuron 111:30-48.e14 (2023). PubMed: 36323321
- Miller JA et al. Distinct Changes in Calpain and Calpastatin during PNS Myelination and Demyelination in Rodent Models. Int J Mol Sci 23:N/A (2022). PubMed: 36499770
- Wu Y et al. Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein. Front Med (Lausanne) 9:811980 (2022). PubMed: 35155498
- Tao Y et al. Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury. J Biol Chem 298:101990 (2022). PubMed: 35490782
- Yang HH et al. Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs. Int J Mol Sci 23:N/A (2022). PubMed: 35163312