Cathepsin L (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cathepsin L run at on SDS-PAGE under reducing conditions?

Under reducing SDS-PAGE, this HEK293-expressed recombinant resolves as two predominant bands: the heavy chain (~25 kDa) and the light chain (~5 kDa) of the processed mature two-chain form, plus a variable proportion of single-chain intermediate (~38 kDa) depending on processing completeness. Under non-reducing conditions, the disulfide-linked two-chain form migrates near 30–32 kDa. Purity is >95% by SDS-PAGE. If you see only the ~38 kDa band, mild acid pre-incubation (pH 5.5, 37°C, 30 min) can drive further autocatalytic maturation before your assay.

What processing form is this recombinant Cathepsin L — procathepsin, single-chain, or mature two-chain?

The recombinant is supplied predominantly as the mature active enzyme, with the propeptide removed via autocatalytic and trans-processing during HEK293 production. You will typically observe a mixture of the mature two-chain form (heavy chain ~25 kDa + light chain ~5 kDa) and some single-chain intermediate (~38 kDa). Both forms carry active-site Cys25/His163 geometry and are enzymatically competent. If your downstream application requires a homogeneous single form, size-exclusion chromatography at pH 5.5 under reducing conditions can enrich the fully processed species.

What substrates does Cathepsin L cleave and what is its preferred cleavage specificity?

Cathepsin L is a papain-family cysteine endopeptidase with a strong preference for hydrophobic residues (Phe, Leu, Val) at the P2 position. The canonical fluorogenic substrate for activity assays is Z-Phe-Arg-AMC (ex/em 355/460 nm), which Cathepsin L cleaves with Km in the low-micromolar range (~10–20 µM under standard conditions). It also processes Z-Leu-Arg-AMC, though with lower efficiency. Physiological substrates include extracellular matrix components (fibronectin, laminin), MHC class II-associated invariant chain, and several pro-hormones. Cathepsin B and L activities can be discriminated using the selective inhibitor CA-074 to block Cathepsin B before measuring residual Z-Phe-Arg-AMC turnover.

What buffer and pH conditions should I use for a Cathepsin L fluorogenic activity assay with Z-Phe-Arg-AMC?

Optimal activity is at pH 5.5–6.0. A standard assay buffer is 50 mM sodium acetate pH 5.5, 4 mM EDTA, 8 mM DTT (or 2 mM TCEP); pre-incubate enzyme in reducing buffer for 10–15 min at 37°C before adding substrate to ensure full active-site thiol reduction. Use Z-Phe-Arg-AMC at 10–50 µM final concentration in a total volume of 100–200 µL. Note: the storage buffer for this recombinant is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — buffer-exchange or dilute ≥10-fold into assay buffer to avoid pH interference.

What starting concentration of recombinant Cathepsin L should I use for IC50 inhibitor profiling?

For IC50 determinations, use 0.5–2 nM active enzyme in assay buffer (50 mM sodium acetate pH 5.5, 4 mM EDTA, 8 mM DTT) with Z-Phe-Arg-AMC at a concentration near the Km (~20 µM). Keeping enzyme well below substrate Km minimises tight-binding artefacts. For jump-dilution or pre-incubation experiments probing covalent inhibitors, a 10-fold pre-incubation concentration (5–20 nM) followed by 10-fold dilution into substrate is practical. Verify linear progress curves for at least 30 min before adding inhibitor series; significant curvature indicates substrate depletion or enzyme instability.

Can I use this recombinant Cathepsin L as a positive control for Western blot with the TPB anti-Cathepsin L antibody?

Yes — this is one of the primary intended uses. Load 10–50 ng of recombinant Cathepsin L per lane on a 12–15% SDS-PAGE gel run under reducing conditions. The matched antibody, RP-CathepsinL (/anti-cathepsin-l-rabbit-polyclonal-antibody), is a rabbit polyclonal raised against human Cathepsin L and is validated for Western blot by Triple Point Biologics. It reliably detects both the ~38 kDa single-chain and the ~25 kDa heavy-chain bands. Starting dilution of RP-CathepsinL for WB is 1:1,000–1:5,000; titrate down if background is high in your lysate matrix.

How much recombinant Cathepsin L should I load as a positive control when validating the RP-CathepsinL antibody by Western blot?

Load 10–100 ng per lane; 25 ng is a practical starting point that gives a clean, quantifiable signal without saturating the film or CCD. Because the recombinant is >95% pure, this lane also serves as a molecular-weight reference: expect a strong band at ~25 kDa (mature heavy chain) and a fainter band at ~38 kDa (single-chain intermediate). Run alongside 20–40 µg of a CTSL-expressing cell lysate (e.g., HeLa or MCF-7) to confirm that RP-CathepsinL (SKU: RP-CathepsinL) detects the endogenous form at the same apparent MW.

How should I store and handle recombinant Cathepsin L to maintain activity — can I refreeze unused aliquots?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is shipped on dry ice. Store at -20°C in the single-use aliquots provided; repeated freeze-thaw cycles progressively reduce specific activity due to thiol oxidation and aggregation. On the day of use, thaw one aliquot on ice, keep at 4°C, and use within 8 hours. For activity assays, pre-reduce the thawed enzyme (8 mM DTT, 10 min, 25°C) before diluting into assay buffer. Avoid diluting to <0.1 nM in the absence of a carrier protein (0.01% BSA helps stabilise low-concentration working stocks).