Kallikrein-14 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-14 on SDS-PAGE or Western blot?

The full-length KLK14 precursor (UniProt Q9P0G3) is 251 amino acids, giving a theoretical MW of ~28 kDa for the unglycosylated polypeptide. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation shifts the apparent MW on reducing SDS-PAGE to approximately 32–36 kDa. You may see a diffuse or doublet band in this range — this is normal and reflects glycoform heterogeneity, not degradation. Under non-reducing conditions the band position is similar, as KLK14 does not form intermolecular disulfides that would alter migration dramatically.

Is this recombinant Kallikrein-14 the zymogen or the mature active form?

This product is supplied as the mature, proteolytically activated form — the pro-peptide has been removed during processing, yielding the catalytically competent serine protease. KLK14 is naturally synthesised as an inactive zymogen requiring cleavage of its short N-terminal pro-sequence to expose the active site. The recombinant supplied here has undergone this activation step, so no additional enterokinase or auto-activation incubation is required before use in activity assays. If your experiment requires the zymogen form specifically, contact us for availability.

What substrates and cleavage specificity does recombinant KLK14 show in activity assays?

KLK14 displays dual trypsin-like and chymotrypsin-like specificity, cleaving after basic residues (Arg, Lys) and aromatic/large hydrophobic residues (Phe, Tyr, Leu). For fluorogenic activity assays, both Boc-Val-Pro-Arg-AMC (trypsin-like substrate) and Suc-Ala-Ala-Pro-Phe-AMC (chymotrypsin-like substrate) have been reported in the literature. A typical starting concentration is 50–200 nM recombinant KLK14 with 100–500 µM fluorogenic substrate in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Monitor AMC release at Ex/Em 360/460 nm. Titrate enzyme concentration within this range to confirm linearity before calculating kinetic parameters.

What buffer conditions are optimal for KLK14 serine protease activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is directly compatible with most activity assay setups; simply dilute into your assay buffer to reduce glycerol to ≤1%. KLK14 activity is optimal between pH 7.0–8.5. Include 0.01–0.05% BSA or Tween-20 to reduce surface adsorption at low enzyme concentrations. Avoid EDTA above 1 mM as it may affect cofactor-dependent buffer components; KLK14 itself is not a metalloprotease and does not require Ca²⁺. PMSF and other serine protease inhibitors will abolish activity and should be excluded from assay buffers.

What starting concentration of recombinant Kallikrein-14 should I use for inhibitor IC50 assays?

For IC50 determinations, use enzyme at or near its Km for your chosen substrate — typically 50–100 nM recombinant KLK14 with a fluorogenic substrate at ~Km concentration (empirically determined but often 100–300 µM for AMC substrates). Working near Km minimises tight-binding artefacts and ensures the Cheng–Prusoff correction for Ki remains valid. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate. Include a PMSF positive-inhibition control and a no-inhibitor baseline. Confirm that substrate conversion remains below 20% over the assay window to maintain initial-rate conditions.

Can I use recombinant Kallikrein-14 as a positive control for Western blot with the matched rabbit polyclonal antibody?

Yes — this recombinant is specifically matched to SKU RP-Kallikrein14, our rabbit polyclonal anti-Kallikrein-14 antibody (/anti-kallikrein-14-rabbit-polyclonal-antibody), and the two are validated together for Western blot. Load 20–50 ng of recombinant KLK14 per lane alongside your cell or tissue lysate. At this loading you should obtain a clean band at ~32–36 kDa (glycosylated form) with RP-Kallikrein14 at a 1:1,000–1:2,000 dilution. This pairing is particularly useful for antibody titration experiments and for confirming that a band in your lysate migrates at the expected position.

How much recombinant Kallikrein-14 should I load for Western blot as a positive control?

Load 20–50 ng per lane as a starting point when using RP-Kallikrein14 (SKU). This amount reliably produces a detectable band at ~32–36 kDa on standard PVDF or nitrocellulose membranes without overwhelming the antibody signal. If you are using a secondary HRP conjugate with enhanced chemiluminescence, start at 20 ng and adjust upward only if signal is weak. Avoid loading >100 ng, as excess recombinant can create streaking artefacts and make accurate MW comparison with endogenous bands in adjacent lysate lanes difficult.

How should I store and handle recombinant Kallikrein-14 to preserve enzymatic activity?

Store at -20°C in the single-use aliquots provided. The buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated to stabilise the enzyme through freeze-thaw; however, repeated freeze-thaw cycles measurably reduce specific activity and should be avoided. On the day of use, thaw one aliquot on ice, dilute into pre-chilled assay buffer, and keep on ice throughout the experiment. For dilutions below ~10 nM, add carrier protein (0.1% BSA) to prevent adsorptive loss on tube walls. Aliquots are stable for up to 12 months at -20°C when stored correctly.