Kallikrein-5 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Kallikrein-5 on SDS-PAGE or Western blot?

The KLK5 precursor (293 aa) has a predicted molecular weight of ~32 kDa, but the HEK293-expressed recombinant typically migrates at 35–40 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. The mature, processed form runs slightly lower (~30–33 kDa) if the pro-peptide has been removed. Expect a diffuse or doublet band in some lots, which is characteristic of heterogeneous glycoforms from mammalian expression. Purity is confirmed at >95% by SDS-PAGE prior to release. If you are running a Western blot positive control alongside our matched antibody RP-Kallikrein5, load the 35–40 kDa region as your primary reference.

Is this recombinant Kallikrein-5 the pro-enzyme or the active mature form?

This product is supplied as the active enzyme form. The pro-peptide has been removed during processing, yielding the mature trypsin-like catalytic domain capable of cleaving substrates directly without further activation. This is distinct from the full-length pre-pro-KLK5 precursor (293 aa). Because the correctly folded active site is preserved through HEK293 mammalian expression, it is suitable for fluorogenic substrate assays, kinetic parameter determinations (Km, kcat), and inhibitor IC50 studies without an additional activation step. If your experiment specifically requires the zymogen form, this product is not appropriate.

What substrates does Kallikrein-5 cleave and which fluorogenic peptide substrate should I use for activity assays?

KLK5 is a trypsin-like serine protease that cleaves after Arg and Lys residues. The most widely used fluorogenic substrate in the literature is Boc-Val-Pro-Arg-AMC, though Tos-Gly-Pro-Arg-AMC is also reported. For a standard activity assay, use 100–400 µM substrate in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20 at 37°C; monitor AMC release at Ex/Em 360/460 nm. Endogenous substrates include desmoglein-1, corneodesmosin, and profilaggrin, relevant to its role in epidermal desquamation and inflammatory skin barrier research.

What buffer conditions are optimal for Kallikrein-5 activity assays and is the storage buffer compatible?

KLK5 shows peak trypsin-like activity between pH 7.5–8.5. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible for direct dilution into assay buffer without buffer exchange. For activity assays, dilute into 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20; the glycerol carryover at working concentrations is negligible. Avoid phosphate buffers at high concentrations, as they can reduce serine protease activity. DTT or reducing agents are generally not required and may be omitted unless your assay design specifically calls for them.

What starting concentration of recombinant Kallikrein-5 should I use for an IC50 inhibitor assay?

For IC50 determinations, a common starting point is 1–5 nM active KLK5 per reaction well, combined with substrate at or near the Km (typically 100–200 µM Boc-Val-Pro-Arg-AMC). Enzyme concentration should be low enough that substrate depletion remains <10% over the assay window, and high enough to give a robust signal-to-background ratio (S/B ≥ 5). Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before substrate addition to allow equilibrium binding. Titrate enzyme concentration empirically in your specific plate format before committing to a full inhibitor panel.

Can I use recombinant Kallikrein-5 as a positive control for Western blot with the RP-Kallikrein5 antibody?

Yes — this is one of the primary intended uses of this product alongside RP-Kallikrein5 (/anti-kallikrein-5-rabbit-polyclonal-antibody). The recombinant protein and the antibody are validated as a matched pair from the same production pipeline, ensuring band detection is confirmed before shipment. Load 50–200 ng per lane under reducing conditions; the antibody recognizes the denatured epitope and you should detect a band at 35–40 kDa (glycosylated form) or ~30–33 kDa (deglycosylated/processed form). This positive control is particularly useful when validating KLK5 expression in low-abundance tissue lysates where endogenous signal may be weak.

How much recombinant Kallikrein-5 should I load as a Western blot positive control and at what antibody dilution?

Load 50–200 ng of recombinant KLK5 per lane alongside your experimental samples. At 100 ng, a clear band at 35–40 kDa is consistently detectable. For RP-Kallikrein5, start at a 1:500–1:2000 dilution in 5% non-fat milk or BSA in TBST for overnight incubation at 4°C; titrate to your chemiluminescence system. The matched antibody has been validated for Western blot, so signal at the expected MW in the recombinant lane serves as both a loading reference and an antibody performance benchmark. Refer to the RP-Kallikrein5 datasheet for IHC antigen retrieval conditions.

How should I handle, aliquot, and store recombinant Kallikrein-5 to preserve enzymatic activity?

Upon receipt, briefly centrifuge the vial and store at -20°C in single-use aliquots — the supplied 10% glycerol provides cryoprotection. Avoid repeated freeze-thaw cycles, as these progressively reduce serine protease activity; plan aliquot sizes based on your typical per-experiment usage. On the day of use, thaw on ice and dilute in assay buffer immediately before use; do not leave the diluted enzyme at room temperature for extended periods. Working stocks can be held at 4°C for up to 48 hours with modest activity loss. Endotoxin is confirmed <0.1 EU/µg by LAL assay, making this suitable for cell-based assays sensitive to LPS contamination.