Cathepsin C (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-CathepsinC
In stock
- SKU
- REC-CathepsinC
Target Overview
Cathepsin C (gene: CTSC; UniProt P53634) is a lysosomal cysteine protease and the founding member of the dipeptidyl peptidase subfamily (EC 3.4.14.1). The 463-amino-acid precursor is processed in the lysosome to yield a mature enzyme that functions as a homotetramer of four heavy-chain/light-chain heterodimers. This recombinant form is expressed in HEK293 cells, a mammalian expression system that supports the glycosylation and processing events relevant to the native human enzyme, making it suitable for biochemical and cell-free assays that require a properly folded, active protease. The enzyme displays broad dipeptidyl peptidase activity, cleaving dipeptide units from the free amino termini of peptide and protein substrates. Substrate tolerance is wide, accommodating both polar and hydrophobic residues, with the constraints that proline cannot occupy the P1 position and arginine cannot occupy the P2 position. In addition to its exopeptidase mode, Cathepsin C can act as an endopeptidase under defined conditions. A key research application is the in vitro activation of granule-associated serine proteases: Cathepsin C is required for the N-terminal processing and activation of neutrophil elastase, cathepsin G, and granzymes A and B. This makes the recombinant protein a practical tool for reconstitution experiments investigating innate immune effector pathways. Researchers use this recombinant to measure enzymatic activity against fluorogenic dipeptide substrates (e.g., Gly-Arg-AMC or Ala-Phe-AMC), to establish IC50 values for small-molecule inhibitors, and to generate a defined antigen for antibody validation. For the latter application, this recombinant can be paired directly with the Triple Point Biologics matched anti-Cathepsin C antibody (RP-CathepsinC), providing a validated positive control for Western blot workflows.
Background
Applications
- Fluorogenic dipeptide substrate cleavage assay (e.g., H-Gly-Arg-AMC or H-Ala-Phe-AMC) to measure specific activity and determine Km and kcat
- Small-molecule inhibitor IC50 determination using continuous fluorescence assay format
- Cell-free reconstitution of granzyme A/B or neutrophil elastase activation by N-terminal dipeptide removal
- Antibody validation positive control for Western blot using the matched Triple Point Biologics anti-Cathepsin C antibody (RP-CathepsinC)
- ELISA standard curve antigen for quantification of Cathepsin C in biological samples
- Selectivity profiling of cysteine protease inhibitor panels across Cathepsin B, C, L, and S
- Substrate specificity mapping using positional scanning synthetic combinatorial libraries (PS-SCL)
References
- Gao W et al. CTSC-RAB38 Potentiates Responsiveness to PD-1 Blockade in Esophageal Squamous Cell Carcinoma. Genomics Proteomics Bioinformatics. 2026. doi:10.1093/gpbjnl/qzag048. PMID: 42316836.
- Zhao J et al. Integrative Transcriptomics and Machine Learning Identify Macrophage-Associated Biomarkers in Hypertrophic Cardiomyopathy. Int J Mol Sci. 2026. doi:10.3390/ijms27115102. PMID: 42278626.
- Ye W. Identification and Validation of Oxidative Stress-Related Candidate Biomarkers for Preeclampsia via WGCNA and Single-Cell Transcriptomics. Am J Reprod Immunol. 2026. doi:10.1111/aji.70241. PMID: 42030320.
- Anderson RA et al. Tissue-Specific Expression of the EWSR1::FLI1 Fusion Protein Identifies col2a1a-Positive Cells as a Source of Ewing Sarcoma-like Tumors in Zebrafish. Int J Mol Sci. 2026. doi:10.3390/ijms27073131. PMID: 41977317.
- Kosek S et al. Targeted proteomic profiling of serum and CSF reveals CASP-8 as a candidate biomarker in anti-NMDAR encephalitis. J Neuroimmunol. 2026. doi:10.1016/j.jneuroim.2026.578927. PMID: 41962323.
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 | Lysosome |
Frequently Asked Questions
What molecular weight bands should I expect for recombinant Cathepsin C on SDS-PAGE or Western blot?
Under reducing SDS-PAGE conditions, expect two bands corresponding to the processed heavy chain (~24 kDa) and light chain (~6 kDa) subunits, which together constitute the mature heterodimer. Under non-reducing conditions the disulfide-linked heterodimer runs near ~30 kDa. The unprocessed ~52 kDa single-chain precursor should be absent or minimal, since this HEK293-expressed preparation undergoes lysosomal-type processing. If you see a dominant 52 kDa band, the enzyme may have lost activity — check processing by activity assay before use.
What is the processing state of recombinant Cathepsin C — is it the proenzyme or the active mature form?
This preparation is the mature, active form. HEK293 expression supports the endosomal/lysosomal processing events that cleave the 463-aa precursor into the heavy-chain (~24 kDa) and light-chain (~6 kDa) subunits. In solution, four heavy/light-chain heterodimers self-assemble into the native homotetramer (~120 kDa native MW). Unlike bacterially expressed Cathepsin C, no separate activation step is required — the enzyme ships in an active state, which you can verify immediately with a dipeptidyl AMC substrate assay.
What substrate should I use to measure recombinant Cathepsin C dipeptidyl peptidase activity in vitro?
Gly-Phe-AMC (also written H-Gly-Phe-AMC) is the standard fluorogenic substrate for Cathepsin C dipeptidyl peptidase activity; cleavage releases the AMC fluorophore (Ex 380 nm / Em 460 nm). Use it at 100–200 µM in 100 mM sodium acetate pH 5.5, 1 mM EDTA, 5 mM DTT. Start with 10–50 ng of this recombinant per 100 µL reaction. Note: proline cannot occupy P1 and arginine cannot occupy P2, so avoid substrates carrying those residues at those positions.
What is the optimal assay buffer and pH for Cathepsin C activity assays with this recombinant?
Peak dipeptidyl peptidase activity occurs between pH 5.0–6.0, consistent with its lysosomal origin. A practical working buffer is 100 mM sodium acetate pH 5.5, 1 mM EDTA, 5 mM DTT. Pre-reduce the enzyme for 10 minutes at room temperature with DTT before adding substrate to ensure free active-site cysteines. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) should be diluted at least 1:5 into assay buffer to avoid pH interference from the Tris-HCl.
How do I determine a starting concentration of recombinant Cathepsin C for inhibitor IC50 studies?
Run a substrate titration first to identify the Km for your chosen substrate (typically ~50–150 µM for Gly-Phe-AMC at pH 5.5). For IC50 assays, use enzyme at the lowest concentration giving a robust linear signal — typically 10–25 ng per 100 µL reaction — and set substrate at or near Km to keep Ki/IC50 relationships interpretable via the Cheng-Prusoff equation. Confirm the signal-to-background ratio is ≥5-fold before running compound titrations. Blanks should contain all assay components except enzyme.
Can I use Cathepsin C (Recombinant) as a positive control for Western blot with the matched TPB antibody RP-CathepsinC?
Yes — this is the intended use case. RP-CathepsinC (/anti-cathepsin-c-rabbit-polyclonal-antibody) was raised and validated against the same human Cathepsin C protein. Load 20–50 ng of this recombinant per lane under reducing conditions; the antibody detects the heavy-chain band at ~24 kDa. Because the antigen and antibody originate from the same production pipeline, this pairing eliminates the ambiguity about whether a faint signal on your experimental samples reflects low expression or antibody incompatibility.
How much recombinant Cathepsin C should I load for a Western blot positive control lane, and at what antibody dilution?
20–50 ng of recombinant Cathepsin C per lane is sufficient for a clean positive control signal under reducing SDS-PAGE. Run it alongside your cell lysate lanes. For RP-CathepsinC, a starting primary antibody dilution of 1:1,000–1:2,000 in 5% non-fat milk/TBST is a reliable starting point; the ~24 kDa heavy-chain band is the primary detection target. If background is high, switch blocking to 5% BSA/TBST, which is often cleaner for anti-cysteine protease antibodies on nitrocellulose.
How should I store and handle recombinant Cathepsin C to preserve activity over time?
Store at -20°C in single-use aliquots. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides cryo-protection, but repeated freeze-thaw cycles progressively denature the active-site cysteine and reduce specific activity — even two extra cycles can cause measurable loss. On the day of use, thaw one aliquot on ice, dilute gently into pre-chilled assay buffer, and discard any remainder. Do not store at 4°C for more than 24 hours. Properly stored aliquots retain activity for up to 12 months from the date of receipt.
Validation imagery coming soon
Western blot validation figures for REC-CathepsinC 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.