Calpain-3 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-Calpain3
In stock
- SKU
- REC-Calpain3
Target Overview
Calpain-3 (UniProt P20807; gene CAPN3) is the 821-amino-acid, muscle-enriched member of the calpain family of calcium-regulated, non-lysosomal cysteine proteases (EC 3.4.22.54). Unlike ubiquitously expressed calpains-1 and -2, Calpain-3 contains three insertion sequences (IS1, IS2, and NS) absent from other family members, which confer autolytic regulation and underlie its skeletal-muscle-predominant expression pattern. This recombinant protein is produced in HEK293 cells, an expression system that supports the mammalian post-translational processing and folding required for enzymatic competence in cysteine proteases of this class. In biochemical settings, researchers use this recombinant to characterise calcium-concentration-dependent protease activity, define cleavage site specificity against defined peptide or protein substrates, and establish dose-response curves for small-molecule calpain inhibitors. Documented substrates include CtBP1 (cleaved at His-409) and p53/TP53, the latter degraded in a proteasome-independent manner in concert with USP25. These substrate relationships make this reagent directly applicable to studies of transcriptional co-repressor processing and tumour-suppressor turnover. For teams running antibody validation workflows, this recombinant serves as a well-defined positive control for Western blot and immunohistochemical detection. It pairs directly with the Triple Point Biologics anti-Calpain-3 polyclonal antibody (RP-Calpain3), allowing researchers to confirm band identity, optimise antibody titration, and establish specificity against the full-length 821-residue sequence in a single-reagent format. The HEK293 expression background also means the preparation is suitable for pull-down and co-immunoprecipitation experiments designed to identify novel Calpain-3 interactors under defined calcium conditions.
Background
Applications
- Calcium-dependent cysteine protease activity assay using fluorogenic peptide substrates (e.g., Suc-LLVY-AMC)
- Inhibitor IC50 determination for small-molecule calpain inhibitor profiling
- Substrate cleavage validation: CtBP1 (His-409) and p53/TP53 processing in cell-free systems
- Western blot positive control and antibody titration standard (pairs with RP-Calpain3)
- Immunohistochemistry antibody validation using recombinant protein-coated controls
- Pull-down and co-immunoprecipitation assays to identify Calpain-3 binding partners under defined [Ca²⁺] conditions
- Autolytic processing studies: time-course SDS-PAGE analysis of IS1/IS2/NS insertion-dependent self-cleavage
- Mass spectrometry-based substrate identification in skeletal-muscle protein lysate spiking experiments
References
- Wang Z et al. Proteomic insights into azoospermia: protein differences in testicular tissue between non-obstructive and obstructive azoospermia patients. Asian J Androl. 2026. doi:10.4103/aja2025114. PMID: 42210639
- Banerjee S et al. Inflammation-Linked Muscle Atrophy in Limb Girdle Muscular Dystrophy R1 (LGMDR1): Insights into Disease Mechanisms. Curr Issues Mol Biol. 2026. doi:10.3390/cimb48040361. PMID: 42042021
- Cui W et al. Protease-mediated PRC1 dissociation promotes H2AK119ub remodeling during stress responses. EMBO J. 2026. doi:10.1038/s44318-026-00729-9. PMID: 41813846
- Severa G et al. French National Protocol for Diagnosis and Care of Calpainopathy (LGMD R1/LGMD D4): consensus guidelines for clinical practice. Orphanet J Rare Dis. 2026. doi:10.1186/s13023-026-04279-5. PMID: 41761360
- Rahmuni Y et al. Implementing a Tiered Genetic Testing Strategy for Muscular Dystrophies in Morocco: From Targeted Assays to Exome Sequencing. Mol Genet Genomic Med. 2026. doi:10.1002/mgg3.70192. PMID: 41742649
Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Cytoplasm; Ca²⁺-activated |
Frequently Asked Questions
What is the expected molecular weight of recombinant Calpain-3 on SDS-PAGE?
Full-length human Calpain-3 (821 aa; UniProt P20807) has a predicted molecular weight of ~94 kDa. On reducing SDS-PAGE, the recombinant produced in HEK293 cells typically resolves as a predominant band near 94 kDa, consistent with the full-length precursor. Autolytic processing can generate fragments of ~58 kDa (C-terminal catalytic core) and ~36 kDa (N-terminal fragment); depending on calcium exposure during handling, you may observe these minor bands. Purity is confirmed at >95% by SDS-PAGE prior to release.
Does recombinant Calpain-3 undergo autolytic processing and which isoform is active?
Yes. Calpain-3 autolytically cleaves itself in a calcium-dependent manner, most prominently within its unique IS2 insertion. The full-length ~94 kDa form is considered the zymogen-like precursor; calcium activation triggers autolysis to yield an active ~58 kDa catalytic fragment. This recombinant is supplied as the full-length form with detectable enzymatic activity, allowing you to study both the intact enzyme and calcium-induced autolytic events in a single preparation. Minimize calcium exposure during aliquoting to preserve the full-length species if that is your experimental requirement.
What substrates can I use to measure Calpain-3 protease activity in a fluorescence assay?
For fluorometric activity assays, the fluorogenic peptide Suc-LLVY-AMC (50–200 µM) is a widely used calpain substrate and provides a reliable signal with Calpain-3 at 0.5–2 µg per reaction. Protein substrates with documented Calpain-3 cleavage sites include CtBP1 (cleaved at His-409) and p53/TP53. Assay buffer should contain 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1–5 mM CaCl₂; omitting CaCl₂ or including 5 mM EGTA serves as a negative control to confirm calcium dependence of the observed activity.
What buffer conditions optimize Calpain-3 activity and how do I set up a calcium titration?
Calpain-3 activity peaks in 50 mM Tris-HCl pH 7.5, 150 mM NaCl with free Ca²⁺ concentrations between 0.1 and 1 mM. For a calcium titration, buffer the free Ca²⁺ with EGTA/CaCl₂ mixtures using a tool such as MaxChelator to hit target free concentrations of 0.01, 0.1, 0.3, 1, and 5 mM. Include 5 mM DTT or 2 mM TCEP to maintain the active-site Cys105 in its reduced form. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with direct dilution into assay buffer; spin the diluted enzyme at 10,000 × g for 2 min before use to remove any aggregates.
What concentration of recombinant Calpain-3 should I use as a starting point for IC50 inhibitor assays?
A standard starting point for IC50 determinations is 1–2 µg recombinant Calpain-3 per 100 µL reaction (~130–260 nM based on ~94 kDa MW). Pre-incubate enzyme with the inhibitor for 15–30 min at room temperature in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT) before initiating the reaction by adding CaCl₂ and substrate. Include a vehicle-only control and a known calpain inhibitor such as MDL-28170 or calpeptin as reference compounds to bracket your IC50 curve and verify assay window quality before testing novel compounds.
How much recombinant Calpain-3 should I load for a Western blot positive control?
Load 50–200 ng per lane for a clean, non-saturated band at ~94 kDa when probing with anti-Calpain-3 antibody (SKU: RP-Calpain3). At 50 ng the band is readily detectable with standard ECL; at 200 ng autolytic fragments (~58 kDa, ~36 kDa) become visible, which can be useful for verifying antibody cross-reactivity across processed forms. Run the recombinant alongside your cell lysate in adjacent lanes so you have a migration reference and a confirmed-positive control on the same blot.
Can I use this recombinant as a positive control to validate the RP-Calpain3 antibody on Western blot?
Yes, and this is the primary cross-use case between these two catalog items. The matched rabbit polyclonal antibody (SKU: RP-Calpain3; see /anti-calpain-3-rabbit-polyclonal-antibody) was validated against recombinant Calpain-3 produced in HEK293 cells — the same expression system as this recombinant — so band identity is unambiguous. Load 50–100 ng of this recombinant as a defined positive control alongside your experimental lysates. This approach is particularly useful when establishing the antibody in a new tissue or cell type where endogenous Calpain-3 expression may be low or absent.
How should I store and handle recombinant Calpain-3 to maintain activity after arrival?
Upon receipt, centrifuge briefly at 500 × g, then aliquot into single-use volumes on dry ice and store at -20°C. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the enzyme through freeze-thaw, but repeated cycling will degrade activity. Avoid introducing CaCl₂ into stock aliquots — calcium triggers autolysis that will reduce full-length protein over time. For dilutions, use storage buffer supplemented with 1 mg/mL BSA for working concentrations below 50 µg/mL. Shelf life is 12 months from date of manufacture when stored continuously at -20°C.
Validation imagery coming soon
Western blot validation figures for REC-Calpain3 will be published here as they are produced in-house.
If you would like to see existing validation data for this antibody before publication, request a sample copy.