Kallikrein-13 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-13 on SDS-PAGE and Western blot?

The full-length KLK13 precursor (277 aa; UniProt Q9UKR3) has a predicted polypeptide MW of ~30 kDa, but this recombinant is produced in HEK293 cells and carries N-linked glycosylation, so the apparent MW on reducing SDS-PAGE typically runs between 35–42 kDa — a smear or broadened band is not unexpected. Under non-reducing conditions, intact disulfide bonds may shift migration further. We recommend using a pre-stained ladder spanning 25–75 kDa and running alongside the matched antibody positive control experiment for unambiguous identification.

Is this recombinant Kallikrein-13 the mature active form or the full-length zymogen precursor?

This product is supplied as the mature, active enzyme form. Like all tissue kallikreins, KLK13 is synthesized with a signal peptide and a short propeptide that must be cleaved for activation. The recombinant is processed during HEK293 expression and purification to yield the catalytically competent mature serine protease. The active-site Ser-His-Asp catalytic triad is intact and functional. Researchers designing zymogen activation experiments should note this is not the pro-enzyme form; if a zymogen is required for your model, contact us to discuss availability.

What substrates does recombinant Kallikrein-13 cleave and how do I measure its protease activity in vitro?

KLK13 is a trypsin-like serine protease with preferential cleavage C-terminal to Arg and Lys residues. For fluorometric activity assays, the synthetic substrate Boc-Val-Pro-Arg-AMC (AMC = 7-amino-4-methylcoumarin) is commonly used and provides a robust signal with KLK13 (excitation ~380 nm, emission ~460 nm). Tosyl-Gly-Pro-Arg-pNA is also compatible for colorimetric readouts at 405 nm. Run assays in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20 at 37°C. Confirm baseline activity with a PMSF-inhibited negative control to establish serine protease-specific signal.

What buffer and assay conditions are optimal for recombinant Kallikrein-13 enzymatic activity experiments?

The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is designed for stability, not peak enzymatic activity. For activity assays, dilute the enzyme into a working buffer of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20 immediately before use — the slightly alkaline pH better supports trypsin-like serine protease catalysis. Keep glycerol below 1% in the final assay volume, as higher concentrations can suppress kcat. Include 1 mM DTT only if reducing conditions are confirmed not to affect your downstream readout, since free thiols can interfere with AMC fluorescence baselines.

What starting concentration of recombinant Kallikrein-13 should I use for a fluorometric protease activity assay?

A practical starting point is 5–25 nM recombinant KLK13 with 100–200 µM fluorogenic substrate (e.g., Boc-Val-Pro-Arg-AMC) in a 100 µL reaction volume. This range typically yields a linear velocity window of 30–60 minutes at 37°C before substrate depletion becomes limiting. If you are profiling inhibitor potency (IC50 determinations), fix the enzyme at 10 nM to keep enzyme concentration well below expected Ki values and reduce tight-binding artifacts. Titrate substrate to at least 3× the apparent Km (~50–300 µM range) to establish proper Michaelis-Menten conditions before proceeding to inhibitor dose-response curves.

Can I use recombinant Kallikrein-13 as a positive control for Western blot with the matched TPB antibody RP-Kallikrein13?

Yes — this is one of the primary design rationales for pairing REC-Kallikrein13 with RP-Kallikrein13. The rabbit polyclonal antibody (SKU: RP-Kallikrein13; details at /anti-kallikrein-13-rabbit-polyclonal-antibody) was raised and validated in the same laboratory that produced this recombinant, so antigen–antibody compatibility is confirmed for Western blot. Load 50–200 ng of recombinant protein per lane on a 12% SDS-PAGE gel under reducing conditions. Expect a band at ~35–42 kDa (glycosylated form). This lane serves as both a size reference and antibody specificity control alongside your experimental lysates.

How much recombinant Kallikrein-13 should I load for a Western blot positive control lane?

Load 50–200 ng per lane as a starting range. At 100 ng, RP-Kallikrein13 (SKU: /anti-kallikrein-13-rabbit-polyclonal-antibody) typically produces a strong, clean signal on a standard ECL Western blot with a 1:1000–1:2000 primary antibody dilution, 12% polyacrylamide gel, and PVDF membrane. If your cell lysate lanes contain very high KLK13 expression, reducing recombinant to 50 ng prevents the positive control from saturating the film and obscuring dynamic range. Always run the positive control in a non-adjacent lane when comparing band intensities across samples to avoid signal bleed-over with sensitive detection systems.

How should I store and handle recombinant Kallikrein-13 to preserve enzymatic activity over time?

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) stabilizes the protein during freeze-thaw, but repeated freeze-thaw cycles will progressively reduce specific activity — treat each aliquot as single-use. Once thawed, keep on ice and use within 4–6 hours; do not refreeze. For dilutions below ~50 µg/mL, add carrier protein (0.1% BSA, protease-free grade) to prevent adsorption to tube walls. Purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so the preparation is suitable for cell-based assays without additional endotoxin depletion steps.