Cathepsin V (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cathepsin V run at on SDS-PAGE or Western blot?

The Cathepsin V precursor (334 aa, UniProt O60911) has a predicted mass of ~37 kDa, but the mature two-chain active form — generated after signal-peptide removal and propeptide cleavage — typically resolves as a heavy chain of ~24 kDa and a light chain of ~5 kDa under reducing conditions. On non-reducing SDS-PAGE the two chains may remain disulfide-linked and migrate together near 29 kDa. HEK293-derived glycosylation can add 2–4 kDa, so expect the heavy chain band between 24–28 kDa depending on gel percentage and reducing conditions.

What is the difference between Cathepsin V, Cathepsin L2, and Cathepsin U — is this the same protein?

Yes, all three names refer to the same gene product (CTSV, UniProt O60911). Cathepsin L2 and Cathepsin U are historical synonyms that appeared in the literature before systematic nomenclature was standardised. Cathepsin V is the current recommended name. It is a distinct paralogue from Cathepsin L (CTSL), sharing ~75% amino acid identity but with a notably restricted tissue expression pattern — predominantly thymus, testis, and corneal epithelium — and a slightly narrower substrate preference profile. When searching inhibitor or substrate literature, cross-checking all three names is advisable to avoid missing relevant data.

What fluorogenic substrate and assay buffer should I use to measure recombinant Cathepsin V activity?

Z-Phe-Arg-AMC is the most widely used fluorogenic substrate for Cathepsin V and gives robust signal at substrate concentrations of 10–50 µM. Assay buffer should reflect the lysosomal compartment: 100 mM sodium acetate or sodium phosphate, pH 5.0–5.5, supplemented with 2–5 mM DTT or 5 mM TCEP to maintain the active-site cysteine in a reduced state, and 1 mM EDTA to chelate inhibitory metal ions. Pre-activate the enzyme in assay buffer for 10–15 minutes at 37 °C before adding substrate. Monitor fluorescence at Ex/Em 380/460 nm.

What starting concentration of recombinant Cathepsin V should I use for an in-vitro activity or inhibitor assay?

For a kinetic activity assay with Z-Phe-Arg-AMC, 1–10 nM recombinant Cathepsin V is typically sufficient to generate a linear, measurable fluorescence signal over 30–60 minutes. For inhibitor IC50 determination, keep enzyme concentration at or below the anticipated Ki of your compound to avoid tight-binding artefacts — 1–5 nM is a practical starting point. If you are titrating a covalent or irreversible inhibitor, a pre-incubation step (30 min, room temperature) before substrate addition helps distinguish true IC50 from inactivation rate. Confirm linearity with a no-inhibitor control in each run.

How does pH affect Cathepsin V activity and should I adjust the assay buffer for different experimental contexts?

Cathepsin V is a lysosomal cysteine protease with a pH optimum typically between 5.0 and 6.0 for most peptide substrates. Activity drops sharply above pH 6.5 and is essentially negligible at neutral or alkaline pH. If you are modelling extracellular tumour microenvironment proteolysis (pH ~6.5–6.8), expect reduced but measurable activity — adjust your assay buffer accordingly and include a pH-matched control. For standard enzymatic characterisation, 100 mM sodium acetate pH 5.5 with 2 mM DTT and 1 mM EDTA gives reproducible results. Always equilibrate the enzyme in assay buffer before commencing the reaction.

Can I use TPB recombinant Cathepsin V as a Western blot positive control for the matched anti-Cathepsin V antibody?

Yes. The TPB anti-Cathepsin V rabbit polyclonal antibody (SKU: RP-CathepsinV; see /anti-cathepsin-v-rabbit-polyclonal-antibody) was developed and validated in the same laboratory as this recombinant, making it a reliable positive-control pairing. Load 20–50 ng of recombinant Cathepsin V per lane on a 12–15% SDS-PAGE gel under reducing conditions. The heavy chain should appear at 24–28 kDa (with glycosylation). Use RP-CathepsinV at the manufacturer's recommended dilution (typically 1:500–1:2000 for Western blot). The HEK293 expression system preserves mammalian post-translational modifications, ensuring the epitope context is biologically relevant.

How much recombinant Cathepsin V should I load for a Western blot positive control, and what band should I expect?

Load 20–50 ng per lane as a starting point alongside your cell lysate samples; this amount typically produces a clear, non-saturated band with chemiluminescent or fluorescent detection systems. On a reducing 12% SDS-PAGE, the major band will be the mature heavy chain at ~24–28 kDa (HEK293 glycoforms shift the exact position slightly). A faint band near 5 kDa corresponding to the light chain may be visible with sensitive detection but can run off standard gels — use a 15–18% or Tricine gel if you want to capture it. Pair with RP-CathepsinV (SKU: RP-CathepsinV) for validated detection.

How should I store and handle recombinant Cathepsin V to maintain activity, and what is the shelf life?

Store at -20 °C in single-use aliquots immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilises the protein during frozen storage. Avoid repeated freeze-thaw cycles; each cycle risks partial denaturation and loss of cysteine-protease activity at the active site. Aliquot to working volumes (typically 5–10 µg per tube) before first use. Under recommended conditions, activity is stable for at least 12 months. Before assay, thaw on ice, briefly centrifuge, and pre-reduce in assay buffer with 2–5 mM DTT for 10–15 minutes at 37 °C to fully activate the enzyme.