Cathepsin H (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Cathepsin H on SDS-PAGE or Western blot?

The 335-amino-acid CTSH precursor has a theoretical MW of ~37 kDa, but the HEK293-expressed recombinant runs at approximately 28–30 kDa under reducing SDS-PAGE conditions, reflecting removal of the signal peptide and propeptide to yield the mature two-chain form, plus N-linked glycosylation. The heavy chain runs near 23–25 kDa and the light chain near 5–6 kDa; under non-reducing conditions these migrate as a single ~28–30 kDa species held by a disulfide bridge. Confirm purity is >90% by the dominant band(s) in that region.

Is this recombinant Cathepsin H the single-chain proenzyme or the mature processed form?

The recombinant is the mature, active form, not the 37 kDa pro-cathepsin H precursor. HEK293 expression and processing yield the disulfide-linked heavy-chain/light-chain heterodimer characteristic of the lysosomal enzyme. Because the propeptide is removed, the product is active upon thawing without an additional activation step — unlike some bacterially expressed zymogens that require exogenous processing. If your experiment specifically requires pro-cathepsin H, this product is not appropriate for that application.

What substrates are recommended for a Cathepsin H aminopeptidase activity assay in vitro?

For aminopeptidase activity, Arg-AMC (H-Arg-7-amido-4-methylcoumarin) is the standard fluorogenic substrate, generating a highly sensitive AMC signal (Ex 360 nm / Em 460 nm). Because Cathepsin H retains endopeptidase activity, Z-Phe-Arg-AMC is also widely used to monitor total cysteine protease activity, though it is less selective. A working substrate concentration of 25–100 µM in 50 mM sodium acetate pH 5.5, 2 mM DTT, 1 mM EDTA is a practical starting point for initial Vmax and inhibitor screening experiments.

What assay buffer conditions should I use to maximize recombinant Cathepsin H activity?

Cathepsin H has a pH optimum in the range of 5.0–6.5, consistent with its lysosomal function. A standard activity buffer of 50 mM sodium acetate pH 5.5, 2 mM DTT, and 1 mM EDTA reliably activates the enzyme before assay — pre-incubate the protein in this buffer for 10–15 minutes at 37°C. Avoid prolonged exposure above pH 7.0, which accelerates inactivation. Note the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) keeps the enzyme stable but is not the assay buffer; dilute accordingly just before use.

What starting concentration of recombinant Cathepsin H should I use for IC50 inhibitor assays?

A starting concentration of 1–5 nM active enzyme is typical for IC50 determination with fluorogenic substrates like Arg-AMC at 50 µM. Confirm your specific lot's activity (units/mg) from the certificate of analysis, then titrate down to the lowest concentration giving a robust signal-to-background ratio (≥5:1) in the linear phase of the reaction. Using the minimum active enzyme concentration minimizes tight-binding inhibitor artifacts and keeps substrate depletion below 10% during the kinetic window — a common source of IC50 overestimation with cysteine proteases.

Can I use recombinant Cathepsin H as a Western blot positive control with the matched TPB antibody RP-CathepsinH?

Yes — this is one of the primary intended cross-uses. REC-CathepsinH and RP-CathepsinH are developed and validated together in-house, so the antibody is confirmed to recognize the same recombinant on Western blot. Load 25–50 ng of REC-CathepsinH per lane alongside your cell lysate samples; expect the antibody to detect the mature ~28–30 kDa band (and potentially a faint ~37 kDa pro-form in cell lysates). This pairing also serves as a reliable antibody validation control, useful if you are working under reproducibility guidelines or preparing a publication methods section.

How much recombinant Cathepsin H should I load for Western blot when validating the RP-CathepsinH antibody?

For a clean positive control lane, 25–50 ng per lane on a standard 12% SDS-PAGE gel is sufficient for detection with RP-CathepsinH at the recommended 1:1,000–1:2,000 primary antibody dilution and an HRP-conjugated secondary. If using enhanced ECL, 10–25 ng may be adequate. Start at 50 ng to establish signal, then optimize downward. Running a two-fold dilution series (50, 25, 12.5 ng) on a single gel is an efficient way to confirm linearity and set your baseline before comparing against endogenous lysate expression levels.

How should I store and handle recombinant Cathepsin H to preserve enzymatic activity over time?

Upon receipt, centrifuge briefly and store at -20°C in the supplied single-use aliquots. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — maintains stability for at least 12 months under these conditions. Avoid repeated freeze-thaw cycles; each cycle measurably reduces specific activity in cysteine proteases. On the day of the experiment, thaw one aliquot on ice, dilute into your assay buffer immediately before use, and discard any remainder rather than refreezing. Carrier protein (0.1% BSA) may be added if working at sub-nanomolar concentrations to reduce surface adsorption losses.