Calpain-11 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-Calpain11
In stock
- SKU
- REC-Calpain11
Target Overview
Calpain-11 (CAPN11; UniProt Q9UMQ6) is a member of the classical calpain family of calcium-regulated, non-lysosomal cysteine proteases. The full-length human sequence spans 739 amino acids. This recombinant protein is produced in HEK293 mammalian cells, a system that supports the post-translational folding environment appropriate for a cytoplasmic cysteine protease and reduces the risk of misfolding artifacts that can accompany prokaryotic expression. Calpain-11 catalyzes limited, selective proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. Based on UniProt annotation (Q9UMQ6), the protein localises to cytoplasmic vesicles, secretory vesicles, and the acrosome, consistent with reported expression data placing CAPN11 prominently in testicular tissue. This subcellular distribution has guided its use in studies of male reproductive biology, particularly investigations into nonobstructive azoospermia where CAPN11 has been identified as a candidate transcript by microarray gene expression profiling (Malcher et al., 2013). Researchers use this recombinant in several in vitro contexts: direct enzymatic activity assays in the presence of defined calcium concentrations, small-molecule inhibitor IC50 determinations, and substrate identification experiments using mass spectrometry-compatible cleavage reactions. Because the protein is expressed in a human cell background, it is particularly suited as a positive control antigen in Western blot and immunohistochemistry validation workflows. Researchers validating detection reagents against Calpain-11 can pair this recombinant with the Triple Point Biologics matched antibody (SKU: RP-Calpain11), which is validated for Western blot against human tissue and predicted to cross-react with dog.
Background
Applications
- Calcium-dependent cysteine protease activity assay using fluorogenic peptide substrates (e.g., Suc-LLVY-AMC)
- Small-molecule calpain inhibitor IC50 determination in isoform-selectivity panels
- Substrate identification by in vitro cleavage followed by mass spectrometry analysis
- Positive control antigen for Western blot validation of anti-Calpain-11 detection reagents
- Positive control antigen for immunohistochemistry (IHC) optimisation and antibody titration
- Antibody validation standard paired with Triple Point Biologics RP-Calpain11 antibody
- Binding affinity measurement (SPR or BLI) of candidate calpain-directed inhibitor compounds
- Recombinant protein standard for quantitative ELISA calibration in expression profiling studies
References
- Macqueen DJ. Characterization of the definitive classical calpain family of vertebrates using phylogenetic, evolutionary and expression analyses. Open Biol. 2014;4:130219. doi:10.1098/rsob.130219. PMID: 24718597.
- Malcher A et al. Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis. Fertil Steril. 2013;100(6):1686–1694.e7. doi:10.1016/j.fertnstert.2013.07.1999. PMID: 24012201.
- Mishra NK et al. Sex-Specific Whole-Transcriptome Analysis in the Cerebral Cortex of FAE Offspring. Cells. 2023;12(2):328. doi:10.3390/cells12020328. PMID: 36672262.
- Gainey SJ et al. Exposure to a firefighting overhaul environment without respiratory protection increases immune dysregulation and lung disease risk. PLoS One. 2018;13(8):e0201830. doi:10.1371/journal.pone.0201830. PMID: 30130361.
- Osipovich AB et al. ZFP92, a KRAB domain zinc finger protein enriched in pancreatic islets, binds to B1/Alu SINE transposable elements and regulates retroelements and genes. PLoS Genet. 2023;19(4):e1010729. doi:10.1371/journal.pgen.1010729. PMID: 37155670.
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-11 on SDS-PAGE or Western blot?
The full-length human Calpain-11 (CAPN11; UniProt Q9UMQ6) spans 739 amino acids with a predicted unmodified MW of approximately 82 kDa. On reducing SDS-PAGE, this HEK293-expressed recombinant typically migrates between 85–90 kDa due to glycosylation and other post-translational modifications supported by the mammalian expression system. Purity is >95% by SDS-PAGE. If running a Western blot positive control alongside our matched antibody RP-Calpain11, load 20–50 ng per lane and expect a clean band in that 85–90 kDa window.
Does recombinant Calpain-11 undergo autoproteolytic processing, and will I see multiple bands?
Classical calpains are known to undergo calcium-dependent autolysis, which can generate N-terminally truncated fragments. For Calpain-11 specifically, autolytic processing under elevated Ca²⁺ conditions may produce a shorter catalytic-domain fragment in addition to the intact ~85–90 kDa band. Our preparation is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol without added CaCl₂, which minimizes autolysis during storage. If you observe a secondary band around 55–60 kDa on your gel, this is consistent with known calpain autoproteolytic behavior rather than a purity issue.
What substrates can I use to measure Calpain-11 protease activity in a fluorescence assay?
Calpain-11 is a calcium-activated cysteine protease. For fluorescence-based activity assays, the synthetic fluorogenic substrate Suc-LLVY-AMC (succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin) is the most widely used for calpain-family members and is a practical starting point. Z-FR-AMC can also be used as a secondary option. Excitation at 360 nm / emission at 460 nm. Include 1–5 mM CaCl₂ to activate the enzyme; omitting calcium serves as a clean negative control. A pre-incubation step of 5 minutes with calcium before substrate addition improves signal consistency.
What buffer conditions optimize Calpain-11 activity, and how much calcium is needed?
A standard activity buffer for Calpain-11 is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, with 1–5 mM CaCl₂ added fresh immediately before the assay. Including a reducing agent such as 1–5 mM DTT or 2 mM β-mercaptoethanol is important to maintain the active-site cysteine (Cys105 in the catalytic domain) in a reduced, competent state. pH is critical — activity drops sharply below pH 7.0 or above pH 8.0. Avoid EDTA or EGTA in assay buffers, as these chelate the calcium required for activation.
What starting concentration of recombinant Calpain-11 should I use for an activity assay or inhibitor IC50 study?
For initial activity assays using Suc-LLVY-AMC or similar substrates, 50–200 nM recombinant Calpain-11 in a 100 µL reaction volume is a reasonable titration range. For inhibitor IC50 experiments, fix the enzyme at a concentration that gives a robust, linear signal over your measurement window — typically 50–100 nM. Use substrate concentrations at or below the Km (typically 50–200 µM for AMC-based substrates with calpains) to ensure sensitivity to competitive inhibitors. Run a no-enzyme background and a no-inhibitor vehicle control in parallel for accurate baseline subtraction.
Can I use recombinant Calpain-11 as a positive control for Western blot with the RP-Calpain11 antibody?
Yes — this is one of the primary intended uses of this recombinant. The matched antibody RP-Calpain11 (/anti-calpain-11-rabbit-polyclonal-antibody) was raised and validated in-house against Calpain-11, and compatibility with this recombinant protein is confirmed. Load 20–50 ng of recombinant Calpain-11 per lane alongside your cell or tissue lysates. The antibody should detect a band at ~85–90 kDa. This pairing is particularly useful when establishing the assay in a new cell line or tissue where endogenous Calpain-11 expression is uncertain.
How much recombinant Calpain-11 should I load as a Western blot positive control, and what dilution of RP-Calpain11 antibody should I use?
Load 20–50 ng of recombinant Calpain-11 per lane for a strong, clean positive control band. For the matched rabbit polyclonal RP-Calpain11, a primary antibody dilution of 1:500–1:2000 in 5% non-fat milk or BSA in TBST is a standard starting range for Western blot; optimize within that window based on your detection system (HRP-conjugated secondary, ECL reagent, and exposure time). The recombinant and antibody are from the same in-house workflow, so cross-reactivity on this specific protein is confirmed rather than predicted.
How should I handle, dilute, and store recombinant Calpain-11 to preserve activity over time?
Recombinant Calpain-11 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol as single-use aliquots. Store at -20°C; do not refreeze after thawing. On the day of use, thaw on ice and briefly centrifuge before opening. For working dilutions, use the same Tris-NaCl buffer supplemented with 0.1% BSA as a carrier to reduce adsorption losses at low protein concentrations. Avoid diluting into PBS or phosphate-based buffers, which can alter enzyme behavior at the pH margins. Prepared working dilutions should be used within 4 hours and not re-stored.
Validation imagery coming soon
Western blot validation figures for REC-Calpain11 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.