Kallikrein-9 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Kallikrein-9 on SDS-PAGE or Western blot?

The full-length KLK9 precursor (UniProt Q9UKQ9) is 250 amino acids with a predicted unglycosylated MW of ~28 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation typically shifts the apparent MW to 32–38 kDa on reducing SDS-PAGE — the exact position can vary slightly between gel systems and glycosylation states. Under non-reducing conditions you may observe a slightly higher or altered band due to intramolecular disulfides. We recommend using a broad-range ladder (10–100 kDa) and running a positive-control lane alongside.

Is this recombinant KLK9 supplied as a zymogen or as the mature active form?

This product is supplied as the mature, processed form — not the full-length zymogen. KLK9 is synthesised endogenously as an inactive preproenzyme; the signal peptide and propeptide are removed during secretion and activation. HEK293 expression allows the secretory pathway to carry out signal peptide cleavage, and the recombinant is processed to yield the secreted mature species. Activity is confirmed prior to release. If your experiment specifically requires the zymogen form for activation kinetics studies, contact us — this is the active enzyme preparation.

What substrates and cleavage preferences does Kallikrein-9 have for activity assays?

KLK9 is a chymotrypsin-like serine endopeptidase (EC 3.4.21.-), preferring cleavage C-terminal to aromatic or bulky hydrophobic residues (Phe, Tyr, Leu). For fluorogenic activity assays, Suc-Ala-Ala-Pro-Phe-AMC is a commonly used substrate for chymotryptic KLK family members and serves as a practical starting point. Monitor fluorescence at Ex 360 nm / Em 460 nm. Confirm substrate conversion linearity with 1–5 nM enzyme before scaling up. Note that substrate selectivity data for KLK9 specifically is more limited than for KLK1 or KLK3, so empirical titration is advisable.

What buffer conditions should I use for Kallikrein-9 activity assays?

The protein is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, dilute into a working assay buffer of 50 mM Tris-HCl pH 8.0, 100–150 mM NaCl, 0.01% Tween-20 or 0.1% BSA to reduce non-specific adsorption at low enzyme concentrations. Serine proteases in the KLK family are inhibited by PMSF and DFP — confirm your substrate and buffer are free of serine protease inhibitors. Avoid reducing agents (DTT, β-ME) above 1 mM, which can destabilize disulfide-dependent structure in the active site.

What starting concentration of recombinant KLK9 is recommended for inhibitor IC50 determination?

For IC50 assays, a practical starting point is 2–5 nM KLK9 with a substrate concentration at or below Km (empirically determined; typically 50–200 µM for AMC-based substrates with KLK family members). Running enzyme at sub-Km substrate concentrations minimizes the substrate competition artifact described by the Cheng-Prusoff correction. Confirm that enzyme concentration is well below the Ki of any reference inhibitor used as a positive control. Titrate enzyme first across 0.5–20 nM to identify a linear activity window, then fix enzyme concentration for the inhibitor series.

Can I use recombinant Kallikrein-9 as a Western blot positive control with the matched Triple Point Biologics antibody?

Yes — this recombinant is designed to pair directly with the matched rabbit polyclonal antibody (SKU: RP-Kallikrein9; /anti-kallikrein-9-rabbit-polyclonal-antibody). Loading 20–50 ng of recombinant KLK9 per lane on a standard 12% SDS-PAGE gel under reducing conditions will give a clean, unambiguous band at ~32–38 kDa, providing a reliable positive control for antibody validation, lot-to-lot comparison, and Western blot optimization. This pairing is particularly useful if you are setting up KLK9 detection in a new tissue lysate or confirming antibody specificity before IHC work.

How much recombinant Kallikrein-9 should I load for Western blot positive control with RP-Kallikrein9?

Load 20–50 ng per lane alongside your experimental samples. At 20 ng with the RP-Kallikrein9 antibody (SKU: RP-Kallikrein9) at a 1:1000–1:2000 dilution and standard ECL detection, you should see a strong band at ~32–38 kDa without signal saturation. If you are using a higher-sensitivity detection system (e.g., fluorescent secondary or enhanced chemiluminescence), start at 10 ng and titrate up. Keep the positive control lane consistent across blots so it doubles as a loading reference for cross-experiment comparisons.

How should I handle, dilute, and store recombinant KLK9 to maintain activity long-term?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C in single-use aliquots. Avoid repeated freeze-thaw cycles — each cycle can measurably reduce serine protease activity. Upon first use, thaw on ice, briefly centrifuge, and dilute in your assay buffer containing a carrier protein (0.1% BSA) to prevent adsorption losses, especially below 10 nM. Do not dilute stock aliquots and refreeze. Purity is >90–95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, relevant if you are using this in cell-based assays.