Kallikrein-12 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-12 on SDS-PAGE or Western blot?

The full-length KLK12 precursor (UniProt Q9UKR0) is 248 amino acids, giving a predicted unmodified MW of ~27 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation shifts the apparent MW to approximately 30–35 kDa on reducing SDS-PAGE — consistent with what is observed for the native secreted form. A diffuse or doublet band in this range is normal and reflects glycan heterogeneity, not degradation. Under non-reducing conditions, mobility is similar since KLK12 lacks inter-chain disulfides in its mature form.

What processing form is this recombinant Kallikrein-12 — zymogen or active mature enzyme?

This product is the processed, active mature form of KLK12. The signal peptide and propeptide have been removed, yielding the trypsin-like serine endopeptidase domain that begins after the conserved activation cleavage site. It is supplied as an active enzyme in solution — no additional activation step is required before use in cleavage or kinetic assays. If your experiment specifically requires the zymogen (pro-KLK12), this product is not the appropriate choice; contact us to discuss custom options.

What peptide substrates does Kallikrein-12 cleave and what sequence specificity should I design around?

KLK12 is a trypsin-like serine protease with a preference for cleavage after Arg or Lys at the P1 position. For fluorogenic activity assays, the substrate Boc-Val-Pro-Arg-AMC or the more widely used H-D-Pro-Phe-Arg-pNA (chromogenic) are reported starting points. Z-Phe-Arg-AMC is also cleaved but with lower efficiency. For kinetic parameter determination (Km, kcat/Km), Boc-Val-Pro-Arg-AMC at concentrations ranging from 25 µM to 1 mM in assay buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.01% Tween-20) is a practical substrate series.

What assay buffer conditions work best for Kallikrein-12 activity and kinetic assays?

KLK12 is active in slightly alkaline, low-ionic-strength conditions. A well-tested starting buffer is 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.01% Tween-20 at 37°C. The enzyme is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — dilute into assay buffer immediately before use to reduce glycerol carry-over below 0.5% in the reaction. Avoid EDTA, which does not inhibit this serine protease directly, but do confirm that no DTT is introduced if testing disulfide-containing inhibitors. Pre-warm enzyme to 37°C for 5 minutes before substrate addition.

What starting concentration of recombinant Kallikrein-12 should I use for an IC50 inhibitor screen?

For IC50 determination, use KLK12 at 1–5 nM in the assay well, confirmed to give a linear signal over the measurement window (typically 30–60 min). Set substrate concentration at or near the Km (empirically determined, reported in the range of 100–300 µM for Arg-AMC substrates) so that the assay is in the Michaelis–Menten linear regime and IC50 values approximate Ki. Include a serine protease inhibitor such as PMSF or aprotinin as a positive inhibition control, and a DMSO vehicle control matched to the highest solvent concentration in your compound series (keep DMSO ≤ 1%).

Can I use recombinant Kallikrein-12 as a positive control for Western blot with the matched rabbit polyclonal antibody?

Yes — this recombinant is the recommended positive control for Western blot with the matched rabbit polyclonal antibody (SKU: RP-Kallikrein12; /anti-kallikrein-12-rabbit-polyclonal-antibody). The antibody and recombinant are produced from the same lab and validated as a pair. Load 20–50 ng of recombinant KLK12 per lane alongside your cell lysate samples. Under denaturing, reducing conditions you should detect a band at approximately 30–35 kDa. This lane also confirms antibody lot-to-lot consistency and serves as a molecular weight marker for the glycosylated species in your biological samples.

How much recombinant Kallikrein-12 should I load for Western blot positive control, and what dilution of RP-Kallikrein12 antibody works?

Load 20–50 ng of this recombinant per lane for a clean, non-saturated signal. For the matched RP-Kallikrein12 rabbit polyclonal antibody, a primary antibody dilution of 1:500–1:2000 in 5% non-fat milk/TBST typically works well, though we recommend titrating on your specific membrane and blocking conditions. The recombinant band at ~30–35 kDa will be sharper than endogenous KLK12 in tissue lysates due to the defined glycosylation profile from HEK293 expression. This pairing is particularly useful for validating antibody performance before committing to IHC or tissue microarray experiments.

How should I store and handle recombinant Kallikrein-12 to preserve enzymatic activity over time?

Upon receipt, store at -20°C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Avoid repeated freeze-thaw cycles — each cycle can reduce activity by 10–20% for serine proteases. For short-term use (within 1 week), the enzyme is stable at 4°C. When diluting for assays, prepare working dilutions in assay buffer immediately before use and keep on ice; do not pre-dilute and re-freeze. Under recommended storage conditions, activity is stable for 12 months from the date of receipt. Purity is >90–95% by SDS-PAGE; endotoxin is confirmed <0.1 EU/µg by LAL assay.