Cathepsin D (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-CathepsinD
In stock
- SKU
- REC-CathepsinD
Target Overview
Cathepsin D (UniProt P07339; gene CTSD) is a lysosomal aspartyl endoprotease classified under EC 3.4.23.5. The full-length human precursor spans 412 amino acids and is proteolytically processed in the lysosome into a two-chain mature form consisting of a light chain and a heavy chain held together non-covalently. This recombinant is expressed in HEK293 cells, a mammalian system that supports the glycosylation and pro-peptide processing characteristic of the native enzyme, making it well suited for enzymatic activity studies under physiologically relevant conditions. At acidic pH (optimum ~3.5–5.0), Cathepsin D cleaves a broad range of protein substrates, including denatured hemoglobin and casein in classical activity assays, as well as physiologically relevant targets such as amyloid precursor protein (APP). Researchers use this recombinant to measure acid protease activity in fluorogenic or chromogenic substrate assays, to determine inhibitor IC50 values for pepstatin A and novel small-molecule candidates, and to characterise substrate specificity by mass spectrometry-based cleavage-site mapping. Because Cathepsin D is trafficked to the lysosome via the mannose-6-phosphate receptor pathway, this recombinant also serves as a positive-control antigen for studies probing lysosomal enzyme sorting, autophagic flux, and endolysosomal dysfunction. Researchers validating anti-CTSD antibodies — including the matched Triple Point Biologics antibody (RP-CathepsinD), which has been validated for Western blot — use this recombinant as a defined positive-control standard to confirm band identity at the expected molecular weight and to titrate antibody working concentrations. Species reactivity of the matched antibody has been validated for human and is predicted for mouse, rat, non-human primate, dog, and pig.
Background
Applications
- Fluorogenic acid protease activity assay using substrates such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 at pH 3.5–4.5
- Inhibitor IC50 determination for pepstatin A and small-molecule aspartyl protease inhibitor candidates
- Substrate cleavage-site mapping by incubation with candidate peptides followed by LC-MS/MS identification
- Antibody validation positive control for Western blot using the matched Triple Point Biologics antibody (RP-CathepsinD)
- Lysosomal enzyme trafficking and sorting assay — recombinant as reference standard for mannose-6-phosphate receptor pathway studies
- Autophagic flux measurement — positive control for Cathepsin D activity in lysosomal degradation assays alongside LC3-II and p62 readouts
- Enzyme-linked immunosorbent assay (ELISA) standard curve calibration for quantification of CTSD in biological fluids
References
- Lee N et al. Fucoxanthin enhances AMPK/mTOR-dependent autophagic flux and attenuates ferroptosis in Alzheimer's disease models. Food Funct. 2026. doi:10.1039/d6fo01264g. PMID: 42333463
- Yu Y et al. E-cigarette aerosols induce the hydrolysis of lysosomal glycerophospholipids through PLA2G4A activation initiated by nicotine binding to CHRNA3/α3 nAChR in airway epithelial cells. Autophagy. 2026. doi:10.1080/15548627.2026.2689038. PMID: 42287088
- Wu X et al. Dynorphin B Promotes Autophagy and Cytotoxicity in Thyroid Cancer Cells via the mTORC1-TFE3 Axis. J Biochem Mol Toxicol. 2026. doi:10.1002/jbt.70952. PMID: 42262709
- Xiao G et al. Defect in lysosomal enzyme trafficking and sorting is associated with irreversibility of pulmonary arterial hypertension. Front Cardiovasc Med. 2026. doi:10.3389/fcvm.2026.1763556. PMID: 42257032
- Pérez-Lázaro S et al. Preclinical serum alterations and tissue changes in protein and gene expression of early cerebrospinal fluid-validated biomarkers in scrapie. Vet Res. 2026. doi:10.1186/s13567-026-01759-1. PMID: 42231460
Additional Specifications
| Specific Activity | Active aspartic protease against fluorogenic peptide substrate at acidic pH |
|---|---|
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
| Sequence (aa range) | amino acids 21–412 (full-length pre-pro form) |
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >95% by SDS-PAGE |
| Expression System | HEK293 |
| Molecular Weight (kDa) | ~45 kDa (pro-form) / ~30 + 14 kDa (mature heavy + light chains) |
| Subcellular Localization | Lysosome |
Frequently Asked Questions
What molecular weight bands should I expect for recombinant Cathepsin D on SDS-PAGE or Western blot?
Under reducing SDS-PAGE conditions, expect two bands corresponding to the processed two-chain mature form: the heavy chain at approximately 34 kDa and the light chain at approximately 14 kDa. Under non-reducing conditions, the non-covalent heterodimer may partially resolve differently. The unprocessed single-chain intermediate (~43 kDa) can also appear as a minor species depending on processing efficiency. This recombinant is expressed in HEK293 cells, so N-linked glycosylation contributes to apparent MW; deglycosylation with PNGase F will shift bands downward by roughly 2–4 kDa.
Is this recombinant Cathepsin D the mature two-chain form or the single-chain precursor?
The HEK293 expression system supports lysosomal-like pro-peptide processing, so the predominant species in this preparation is the mature two-chain form (light chain + heavy chain held non-covalently), consistent with the native human enzyme (UniProt P07339). A minor fraction of single-chain intermediate (~43 kDa) may be present. If your experiment requires a strictly homogeneous precursor or a defined single-chain construct, contact us to confirm lot-specific processing data before ordering.
What substrates work best for a Cathepsin D activity assay and what pH should I use?
The standard fluorogenic substrate is the FRET peptide MOCAc-GKPILFFRLK(Dnp)-D-R-NH2, used at 10–20 µM in 100 mM sodium acetate, 200 mM NaCl, pH 3.5–4.0. Classical chromogenic assays use denatured hemoglobin or acid-soluble casein at pH 3.5. Cathepsin D activity is optimal between pH 3.5 and 5.0; activity drops sharply above pH 6.0. Pre-activate the enzyme at 37°C for 5–10 minutes in assay buffer before adding substrate to allow complete auto-activation of any residual single-chain species.
What starting concentration of recombinant Cathepsin D should I use for an IC50 assay with pepstatin A?
A working concentration of 0.5–2 nM recombinant Cathepsin D in 100 mM sodium acetate pH 3.5 is typical for pepstatin A IC50 determinations using the MOCAc fluorogenic substrate. Literature IC50 values for pepstatin A against Cathepsin D fall in the low nanomolar range (~1–10 nM inhibitor). Titrate inhibitor from 0.001 nM to 1 µM in a 10-point, 3-fold dilution series. Verify that substrate conversion remains below 20% at the chosen enzyme concentration to maintain initial-rate kinetics throughout the assay window.
Can I use recombinant Cathepsin D as a positive control for Western blot with the matched TPB antibody RP-CathepsinD?
Yes — this is a primary use case for pairing REC-CathepsinD with RP-CathepsinD. Load 10–50 ng of recombinant protein per lane alongside your cell lysate samples. The rabbit polyclonal (RP-CathepsinD) is validated for Western blot and should detect the heavy chain (~34 kDa) and light chain (~14 kDa) bands under reducing conditions. Because both products originate from the same TPB program, epitope compatibility is guaranteed, making this combination reliable for antibody validation experiments and for confirming positive signal in new cell or tissue lysate systems.
How much recombinant Cathepsin D should I load for a Western blot positive control lane?
10–50 ng per lane is the recommended range when using RP-CathepsinD at a 1:1,000–1:3,000 dilution. Start at 25 ng to establish baseline signal, then titrate up or down based on your film or digital imager sensitivity. Because this preparation is >90% pure by SDS-PAGE, nearly all detected signal will correspond to the mature heavy and light chains, minimizing background interpretation issues. Run it in a dedicated lane adjacent to your experimental lysates so band positions are unambiguous.
How should I store recombinant Cathepsin D and what is its working shelf life after thawing?
Store at -20°C in single-use aliquots in the supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Avoid repeated freeze-thaw cycles, as these progressively reduce enzymatic activity. Once thawed, keep on ice and use within the same working session — ideally within 4–6 hours. Aliquot upon first receipt if your lot size exceeds a single experiment's need. Glycerol at 10% provides cryoprotection; do not dilute the stock below ~5% glycerol unless you plan to use it immediately, as this reduces freeze-thaw stability.
Does recombinant Cathepsin D cleave amyloid precursor protein and can I use it for APP processing studies?
Cathepsin D cleaves amyloid precursor protein (APP) at acidic pH, producing amyloidogenic fragments including Aβ-like peptides — a well-documented activity relevant to Alzheimer's disease research. This HEK293-expressed recombinant retains that activity and is suitable for in vitro APP cleavage assays. Use full-length or truncated APP substrates at pH 4.0–5.0 in acetate buffer, with 0.5–5 nM enzyme and substrate concentrations in the µM range. Confirm cleavage products by SDS-PAGE or mass spectrometry; RP-CathepsinD can be used to verify enzyme presence in the reaction by Western blot.
Validation imagery coming soon
Western blot validation figures for REC-CathepsinD 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.