Kallikrein-11 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Kallikrein-11 on SDS-PAGE or Western blot?

The KLK11 precursor (282 aa; UniProt Q9UBX7) has a predicted unglycosylated MW of ~30 kDa, but because this recombinant is expressed in HEK293 cells and carries native-like N-glycosylation, the apparent MW on reducing SDS-PAGE typically runs between 35–42 kDa. Exact migration varies with glycan heterogeneity. Under non-reducing conditions a slightly higher or diffuse band is common. If you treat the sample with PNGase F before running, you should collapse the band back toward ~30 kDa — a useful internal check that the size shift is glycosylation-dependent.

Which processed form of Kallikrein-11 does this recombinant represent — zymogen, active enzyme, or cleaved chains?

The product is supplied as the active, single-chain enzyme generated after removal of the signal peptide and propeptide from the 282 aa precursor. This corresponds to the catalytically competent form found in secretions. The two inactive chain fragments produced by further auto- or trans-cleavage are not the predominant species in this preparation. If your experiment requires the zymogen form or a specific processing intermediate, this active preparation is not the appropriate starting material — contact us to discuss alternatives.

What substrate should I use to measure Kallikrein-11 activity in a fluorogenic assay?

Bz-Phe-Arg-AMC (benzoyl-Phe-Arg-7-amido-4-methylcoumarin) is the preferred fluorogenic substrate for KLK11. The enzyme shows a marked preference for Arg at the P1 position over Lys, so Arg-AMC-based substrates outperform Lys-AMC equivalents in head-to-head comparisons. Monitor liberation of AMC at Ex 380 nm / Em 460 nm. A practical starting point is 100 µM substrate with 1–5 nM recombinant KLK11 in assay buffer; optimize enzyme concentration to keep reaction progress linear over your measurement window before running inhibitor titrations or IC50 experiments.

What assay buffer conditions are recommended for Kallikrein-11 activity and inhibitor studies?

KLK11 is a serine protease that is active across a broad near-neutral pH range. A standard working buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 (to reduce non-specific surface adsorption) performs reliably. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most activity assays at the dilutions used; glycerol carryover is negligible at ≤0.5% final concentration. Avoid EDTA above 5 mM, though unlike metalloproteinases KLK11 is not zinc-dependent and EDTA at low concentrations does not inhibit activity.

What starting concentration of recombinant Kallikrein-11 should I use for an IC50 inhibitor assay?

For IC50 determinations, start with an enzyme concentration that gives a linear, robust signal without substrate depletion over the assay window — typically 1–5 nM KLK11 with 50–200 µM Bz-Phe-Arg-AMC. Pre-incubate enzyme with inhibitor for 15–30 minutes at 37°C before substrate addition to allow equilibrium binding, particularly for slow-on inhibitors. Verify that less than 10% of substrate is consumed at the assay endpoint to maintain steady-state conditions. If your inhibitor is a standard serine protease mechanism-based inhibitor (e.g., aprotinin, PMSF), include appropriate positive-control wells at each plate.

Can I use recombinant Kallikrein-11 as a positive control on a Western blot with the matched TPB antibody?

Yes — this is a primary validated use case. The matched antibody RP-Kallikrein11 (/anti-kallikrein-11-rabbit-polyclonal-antibody) was raised and validated in the same laboratory against KLK11 protein, making this recombinant the most reliable positive control available for Western blot. Load 20–50 ng of recombinant KLK11 per lane alongside your lysate samples. Under reducing conditions, expect a band at 35–42 kDa (glycosylated) or ~30 kDa post-PNGase F treatment. This lane confirms antibody performance and provides a size reference independent of endogenous expression levels in your cell or tissue samples.

How much recombinant Kallikrein-11 should I load for a Western blot positive control lane?

20–50 ng per lane is a practical starting range with RP-Kallikrein11 at typical working dilutions (1:500–1:2000 on Western blot). If you are titrating antibody concentration or comparing lot-to-lot sensitivity, a two-fold dilution series from 100 ng down to 6.25 ng across adjacent lanes gives a clean reference ladder. Because this recombinant is produced in HEK293 cells and is glycosylated, the band will run slightly higher than a bacterially expressed fragment, which is an important visual distinction from prokaryotic positive controls used in some published protocols.

How should I handle, dilute, and store recombinant Kallikrein-11 to maintain activity?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and stored at -20°C in single-use aliquots. Thaw on ice, briefly centrifuge the tube, and dilute into assay buffer immediately before use. Avoid repeated freeze-thaw cycles — even one additional cycle can measurably reduce specific activity for serine proteases. For short-term working stocks (1–4 hours on ice), add 0.1% BSA as a carrier if working below 10 nM to prevent adsorptive loss to tube walls. Purity is >95% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL, suitable for cell-based assays.