Kallikrein-4 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-4 on SDS-PAGE and Western blot?

The KLK4 open reading frame encodes a 254-amino-acid preproprotein (~28 kDa predicted). Because this recombinant is produced in HEK293 cells, native-like N-linked glycosylation is present, shifting the apparent molecular weight on SDS-PAGE to approximately 32–36 kDa under reducing conditions. A diffuse or doublet band in this range is normal and reflects glycan heterogeneity, not degradation. If you are running a positive control lane alongside immunoprecipitated samples, expect the band to run slightly higher than the calculated sequence mass.

Is this recombinant Kallikrein-4 the active mature form or the full-length zymogen?

This product is supplied as the active, mature form of KLK4, with the N-terminal signal peptide and propeptide removed. The mature protease begins at Ile-21 (after propeptide cleavage) and carries a fully intact trypsin-like catalytic triad (His-41, Asp-91, Ser-182 in mature numbering). Zymogen activation of KLK4 is mediated in vivo by KLK3 (PSA) or by plasmin; purchasing the pre-activated mature form eliminates this activation step and allows direct use in kinetic or inhibitor assays without an additional activation protocol.

What substrates does Kallikrein-4 cleave and which fluorogenic peptide is best for activity assays?

KLK4 is a trypsin-like serine protease that cleaves after basic residues (Arg, Lys). The most widely used fluorogenic substrate is Boc-Val-Pro-Arg-AMC, which gives robust signal at low nanomolar enzyme concentrations. Alternatives include H-D-Val-Leu-Arg-AFC and the generic trypsin substrate Tos-Gly-Pro-Arg-AMC. Read fluorescence at Ex/Em 380/460 nm (AMC) or 400/505 nm (AFC). Known physiological substrates include PAR-1, PAR-2, LEKTI domains, and components of the enamel matrix, making KLK4 relevant to both skin barrier and dental mineralisation research.

What buffer conditions are recommended for Kallikrein-4 activity assays and does the storage buffer interfere?

The protein is shipped in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For fluorogenic peptide assays, dilute into assay buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.01% Tween-20) to keep glycerol ≤1% in the final reaction — higher glycerol can reduce kcat by ~15–20% and interfere with AMC fluorescence. Avoid EDTA or reducing agents such as DTT above 1 mM, as disulfide integrity is critical for correct KLK4 fold. Calcium is not required for KLK4 activity (unlike metalloproteinases).

What starting concentration of recombinant Kallikrein-4 should I use for an IC50 inhibitor assay?

For IC50 determinations, a common starting point is 1–5 nM enzyme with substrate (Boc-Val-Pro-Arg-AMC) at 50–100 µM, which approximates the Km reported in the literature (~80–120 µM for AMC substrates). Keeping enzyme well below substrate concentrations and using the Morrison tight-binding correction is advisable if your inhibitor Ki is expected to fall below 10 nM. Run a substrate titration first to confirm linearity of signal over your assay window. Pre-incubate enzyme with inhibitor for 30 min at 37°C before initiating the reaction with substrate.

Can I use this recombinant Kallikrein-4 as a positive control antigen for Western blot with the matched TPB antibody?

Yes — this is a primary use case for pairing REC-Kallikrein4 with the matched rabbit polyclonal antibody (SKU: RP-Kallikrein4; /anti-kallikrein-4-rabbit-polyclonal-antibody). Load 20–50 ng of recombinant protein per lane on a 12% SDS-PAGE gel. The antibody detects the glycosylated band at ~32–36 kDa. Because both the recombinant and the antibody are produced and validated in the same laboratory, the pairing is confirmed for Western blot detection without the ambiguity that can arise when mismatched reagents from different vendors are combined.

How much recombinant Kallikrein-4 should I load to validate the TPB anti-Kallikrein-4 antibody by Western blot?

For antibody validation purposes, a two-point titration of 10 ng and 50 ng per lane is sufficient to demonstrate linearity of the RP-Kallikrein4 signal and confirm specificity against the single ~32–36 kDa band. Run alongside a lysate lane (e.g., LNCaP or SW780 cells, which express endogenous KLK4) to confirm the antibody detects the endogenous protein at the same apparent molecular weight. This side-by-side format satisfies antibody validation guidelines (e.g., MIQE-adjacent reporting standards) requiring a positive recombinant control matched to the antibody's target.

How should I handle, dilute, and store recombinant Kallikrein-4 to preserve activity over time?

Upon receipt, briefly centrifuge the vial, then aliquot immediately into single-use volumes at -20°C — avoid repeated freeze-thaw, which degrades serine protease activity progressively. For working dilutions, use assay buffer containing 0.01–0.05% BSA as a carrier to prevent adsorption to tube walls at concentrations below 10 µg/mL. Thawed aliquots are stable on ice for 4–6 hours; do not leave at room temperature longer than necessary. If activity drops unexpectedly after storage, run a fluorogenic substrate check (Boc-Val-Pro-Arg-AMC at 100 µM) before attributing loss to the experimental condition.