• Product name
    Calpain Activity Assay Kit
    See all Calpain 1 kits
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
  • Assay time
    2h 00m
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mammals
  • Product overview

    Calpain Activity Assay Kit (ab65308) provides optimized buffers and reagents for a convenient measurement of calpain activity. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). The fluorometric assay 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.

    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca²+, and activated calpain is released into cytosol.


    If additional Ac-LLY-AFC substrate is needed, it can be purchased separately as ab171379.


  • Storage instructions
    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
  • Function
    Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
  • Tissue specificity
  • 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
  • Alternative names
    • Ca2 activated neutral protease
    • Calcium activated neutral proteinase
    • Calcium activated neutral proteinase small subunit
    • Calcium dependent protease small subunit
    • Calcium dependent protease small subunit 1
    • Calcium-activated neutral proteinase 1
    • Calpain 1
    • Calpain 1 large subunit
    • Calpain 1, (mu/I) large subunit
    • Calpain mu type
    • Calpain mu-type
    • Calpain regulatory subunit
    • Calpain small subunit 1
    • Calpain, large polypeptide L1
    • Calpain-1 catalytic subunit
    • Calpain-1 large subunit
    • CAN1_HUMAN
    • CANP
    • CANP 1
    • CANP small subunit
    • CANP1
    • CANPL 1
    • CANPL1
    • CAPN 1
    • CAPN1
    • Cell proliferation inducing protein 30
    • Cell proliferation-inducing gene 30 protein
    • Micromolar Calpain
    • Micromolar-calpain
    • Mu Calpain
    • muCANP
    • muCL
    see all
  • Database links

Associated products


  • Different amounts of positive control (Calpain I) treated with 1 μL of inhibitor (Z-LLY-FMK), background signal subtracted, duplicates; +/- SD.

  • 10e7 Jurkat cells (in 10 mL) were cultured in the absence or presence of 10 μM Camptothecin (CPT) (ab120115) or 10 μg/mL Cycloheximide (CHX) (ab120093) for 4 hours. Pelleted cells were lysed in 0.5 mL of Extraction Buffer and tested directly. 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.



This product has been referenced in:
  • Wolhuter K  et al. Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation. Mol Cell 69:438-450.e5 (2018). Functional Studies . Read more (PubMed: 29358077) »
  • Pahima H  et al. Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities. Oncotarget 9:12825-12841 (2018). Read more (PubMed: 29560113) »

See all 19 Publications for this product

Customer reviews and Q&As

Ab65308 measures overall calpain activity. It will measure activity from any active Calpain isoform in the sample. Calpain I and II are mainly expressed in the central nervous system. So depending on what your sample is, the calpain isoform can differ....

Read More
Calpain activity kit tested in rat's gastrocnemius (fast fibers). 2 conditions: control and 7 days of sepsis.
10mg of muscle was crushed with ultraturax on ice in the extraction buffer of the kit. After a centrifugation (4min, 4°C, 11000rpm) proteins were quantify in supernatant by a Bradford test. The kit was made according to the instructions, samples in duplicate and read at 405/510nm.
the figure explain the % of variation of calpain activity in septic muscle compare to control group.
No difference was observed

Dr. Florine Tissier

Verified customer

Submitted Feb 12 2018

There is ˜50 mM Calcium in the 10X reaction buffer but not in Extraction buffer. The Extraction buffer however is supplied with optimal concentrations of EDTA and EGTA to chelate the calcium to avoid any autoactivation of calpain during the extraction ...

Read More

For the Calpain assay we highly recommend using fresh tissues or tissue samples snap-frozen after collection. The frozen tissue can be ground and then homogenized in the extraction buffer.

One point to note is : Inactive calpain remains a...

Read More

The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.

Immediate freezing of the sample in aliquots and storing ...

Read More

This kit has the substrate bound to the AFC fluorophore.

The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.
Immediate freezing of the sample in aliquots and storing for up to 2 mont...

Read More

I can confirm that for the sample preparation the cells will not need to be trypsinized. They can be scraped from the flask after after adding the extraction buffer and incubated, but keep in mind that this would dilute the sample for the activity assa...

Read More

This Calpain Activity Assay Kit includes Active Calpain I as positive control.

The Calpain protein provided in ab65308 is a native protein. We pack 14.5 ug of Calpain/tube in this kit.

1-10 of 27 Abreviews or Q&A


Sign up