Kallikrein-10 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Kallikrein-10 on SDS-PAGE or Western blot?

The mature, processed form of KLK10 runs at approximately 26–30 kDa under reducing SDS-PAGE conditions. The apparent MW on gel typically exceeds the theoretical ~26 kDa of the core polypeptide due to N-linked glycosylation added during HEK293 expression. Under non-reducing conditions, disulfide-dependent compaction may shift the band slightly. If you are running a Western blot positive control alongside our matched antibody RP-Kallikrein10, load 50–100 ng per lane and expect a diffuse or doublet band in the 26–32 kDa range consistent with glycoform heterogeneity.

What processing form is this recombinant Kallikrein-10 — zymogen or active enzyme?

REC-Kallikrein10 is supplied as the active, mature enzyme following signal peptide and propeptide removal, consistent with the zymogen maturation pattern of kallikrein-related peptidases. It is not a pro-form. The catalytic triad (His-Asp-Ser) is intact and the enzyme is active as supplied. No additional activation step is required before use in activity assays. If your experiment specifically requires the zymogen precursor for maturation studies, contact us — but for substrate cleavage, inhibitor profiling, or antibody validation work, the active form is appropriate.

What substrates does recombinant Kallikrein-10 cleave and what is its protease class?

KLK10 is a secreted serine protease with a chymotrypsin-fold catalytic mechanism. Depending on assay conditions, it can exhibit trypsin-like (cleaving after Arg/Lys) or chymotrypsin-like (cleaving after Phe/Tyr/Leu) substrate preferences. For fluorogenic activity assays, Boc-Val-Pro-Arg-AMC (trypsin-like) and Suc-Ala-Ala-Pro-Phe-AMC (chymotrypsin-like) are commonly used substrates. Run both substrate types alongside a blank (buffer only) to determine which preference predominates under your specific buffer and pH conditions before committing to downstream inhibitor or substrate profiling experiments.

What buffer and pH conditions work best for a Kallikrein-10 activity assay?

REC-Kallikrein10 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, dilute into a working buffer of 50–100 mM Tris-HCl or PBS at pH 7.4–8.0; serine protease activity is generally attenuated below pH 6.5. Include 0.01–0.05% BSA or Tween-20 to minimize surface adsorption at low enzyme concentrations. Avoid EDTA-containing buffers, as divalent cation chelation can indirectly affect assay components. A starting enzyme concentration of 1–5 nM in the assay well is reasonable for initial Km/Vmax determination with AMC-tagged peptide substrates.

What starting concentration of recombinant Kallikrein-10 should I use for an inhibitor IC50 assay?

For inhibitor IC50 determination, use the lowest enzyme concentration that gives a robust, linear fluorescence signal above background — typically 1–5 nM KLK10 in a 100 µL assay volume with substrate at or near the Km (start at 50–200 µM for AMC-tagged substrates). Running enzyme significantly above Km will compress the apparent IC50 of competitive inhibitors, so titrate enzyme and substrate concentrations in a preliminary experiment. Include serine protease inhibitor PMSF (1 mM final) as a positive inhibition control to confirm assay sensitivity before running compound plates.

Can I use recombinant Kallikrein-10 as a Western blot positive control for the RP-Kallikrein10 antibody?

Yes — REC-Kallikrein10 and RP-Kallikrein10 are produced and validated together in the same lab specifically for this purpose. Load 50–100 ng of recombinant protein per lane on a 12–15% SDS-PAGE gel (reducing conditions) and transfer to PVDF or nitrocellulose. The rabbit polyclonal RP-Kallikrein10 is validated for Western blot and should be used at 1:500–1:2,000 depending on your secondary antibody system. The expected band at 26–32 kDa provides a clean reference when troubleshooting low or absent signal in cell lysate or tissue samples. See /anti-kallikrein-10-rabbit-polyclonal-antibody for full validation data.

How much recombinant Kallikrein-10 should I load as a positive control alongside RP-Kallikrein10 for IHC antibody validation?

For Western blot antibody validation alongside RP-Kallikrein10, 50–100 ng per lane is a reliable starting point. If you are establishing a dilution curve to demonstrate antibody linearity — useful for IHC validation papers — load 25, 50, and 100 ng lanes in parallel with a KLK10-positive cell lysate (e.g., LNCaP or MCF-7). This bracketing approach demonstrates dose-dependent signal and confirms the band identity before you commit to IHC optimization. Note that RP-Kallikrein10 is validated for WB (immunohistochemistry (IHC) and immunofluorescence (IF) validation in progress); see the antibody page at /anti-kallikrein-10-rabbit-polyclonal-antibody for species cross-reactivity data.

How should I store and handle recombinant Kallikrein-10 to maintain enzymatic activity over time?

Store REC-Kallikrein10 at -20°C in single-use aliquots as supplied. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated to stabilize active serine proteases during frozen storage. Avoid repeated freeze-thaw cycles — each cycle can reduce specific activity due to partial denaturation. On the day of use, thaw on ice and dilute immediately into assay buffer with a carrier protein such as 0.1% BSA if working below 10 nM. Do not vortex; mix gently by pipette. Properly stored aliquots are stable for at least 12 months from date of receipt.