Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
Product nameCalpain Activity Assay Kit
See all Calpain 1 kits
Sample typeTissue Extracts, Cell Lysate
Assay time2h 00m
Species reactivityReacts with: Mammals, Other species
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
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.
Storage instructionsStore 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
FunctionCalcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
Sequence similaritiesBelongs to the peptidase C2 family.
Contains 1 calpain catalytic domain.
Contains 4 EF-hand domains.
Cellular localizationCytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding.
- Information by UniProt
- Ca2 activated neutral protease
- Calcium activated neutral proteinase
- Calcium activated neutral proteinase small subunit
Different amounts of positive control (Calpain I) treated with 1 μL of inhibitor (Z-LLY-FMK), background signal subtracted, duplicates; +/- SD.
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.
ab65308 has been referenced in 46 publications.
- Wang F et al. Prolonged unfolded protein reaction is involved in the induction of chronic myeloid leukemia cell death upon oprozomib treatment. Cancer Sci 112:133-143 (2021). PubMed: 33067904
- Nogami K et al. Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. Hum Mol Genet 30:1006-1019 (2021). PubMed: 33822956
- Tian X et al. Inhibiting calpain 1 and 2 in cyclin G associated kinase-knockout mice mitigates podocyte injury. JCI Insight 5:N/A (2020). PubMed: 33208557
- Chen L et al. The role of desmin alterations in mechanical electrical feedback in heart failure. Life Sci 241:117119 (2020). PubMed: 31794771
- Li M et al. ACYP2 contributes to malignant progression of glioma through promoting Ca2+ efflux and subsequently activating c-Myc and STAT3 signals. J Exp Clin Cancer Res 39:106 (2020). PubMed: 32517717