Kallikrein-15 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-15 on SDS-PAGE and Western blot?

The KLK15 precursor encodes 256 amino acids, giving a theoretical unglycosylated MW of ~28.8 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation is expected to shift the apparent MW on SDS-PAGE to approximately 32–36 kDa under reducing conditions — consistent with the range reported for other HEK293-expressed KLK family members. Run alongside a broad-range ladder and do not expect a tight band at the unglycosylated theoretical mass. Deglycosylation with PNGase F will collapse the band toward ~28–29 kDa if confirmation is needed.

What processed form is recombinant Kallikrein-15 supplied in — zymogen or active enzyme?

The product is supplied as the active, processed enzyme, not the full-length 256-aa zymogen. Signal peptide cleavage occurs during HEK293 secretion, and the propeptide is removed to yield the mature serine protease domain. The result is an active-form protein without the need for exogenous activation steps. If your experiment requires the zymogen as a comparator, activation state should be considered in experimental design. Single-use aliquots are recommended to prevent autolytic degradation over repeated freeze-thaw cycles.

What substrates can I use to measure Kallikrein-15 serine protease activity in a fluorescence assay?

KLK15 is predicted to have trypsin-like activity based on its conserved catalytic triad and family homology, so fluorogenic substrates with Arg or Lys at the P1 position are the most logical starting point. Boc-Val-Pro-Arg-AMC and H-D-Pro-Phe-Arg-AMC are commonly used for trypsin-like KLKs and represent practical first choices. Note that the definitive endogenous substrate of KLK15 has not been established, so synthetic substrate Km and kcat values should be interpreted as activity confirmation tools rather than physiologically calibrated parameters. Include a no-enzyme blank and a heat-inactivated control in each run.

What buffer conditions are recommended for Kallikrein-15 activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most activity assay formats without dilution-driven pH shifts, provided the enzyme is diluted at least 1:10 into assay buffer so that final glycerol is ≤1%. A standard assay buffer of 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.01% Tween-20 works well for fluorogenic AMC-substrate assays. Avoid EDTA-containing buffers; although KLK15 is a serine protease and not strictly zinc-dependent, divalent cation chelators can affect assay consistency. Pre-warm enzyme and substrate to 37°C before combining.

What starting concentration of recombinant Kallikrein-15 should I use to establish an IC50 for a serine protease inhibitor?

For IC50 determinations, a starting enzyme concentration of 1–5 nM active KLK15 is a reasonable range with AMC-based fluorogenic substrates at substrate concentrations near the apparent Km (~50–200 µM, depending on substrate). Use at least 1 nM to maintain a signal-to-noise ratio adequate for curve fitting, but keep enzyme well below inhibitor concentration to satisfy tight-binding assumptions. Pre-incubate enzyme with inhibitor for 15–30 minutes at 37°C before substrate addition. Confirm linearity of the progress curve under chosen conditions before committing to the full inhibitor dilution series.

Can I use recombinant Kallikrein-15 as a positive control for Western blot with the matched TPB antibody RP-Kallikrein15?

Yes — this is a primary intended use case. REC-Kallikrein15 and the matched rabbit polyclonal antibody RP-Kallikrein15 (/anti-kallikrein-15-rabbit-polyclonal-antibody) are validated together from the same production lot for Western blot compatibility. Load 25–100 ng of REC-Kallikrein15 per lane under reducing conditions; the glycosylated band at ~32–36 kDa should be cleanly detected. This provides a reliable size marker and signal reference when probing tissue lysates or conditioned media for endogenous KLK15, and removes ambiguity about whether a negative result reflects absent protein or antibody failure.

How much recombinant Kallikrein-15 should I load for a Western blot positive control, and what band should I expect?

Load 50 ng as a starting point; this typically yields a clear, well-defined band with RP-Kallikrein15 at standard ECL exposure times. If your secondary antibody or detection system is highly sensitive, 25 ng may suffice and will reduce background bleed into adjacent lanes. Expect the primary band at approximately 32–36 kDa due to HEK293-derived glycosylation. A faint lower band near 28–29 kDa may appear and likely reflects partially deglycosylated or unglycosylated species — this is normal and does not indicate degradation. Run under reducing conditions with fresh β-mercaptoethanol or DTT for consistent results.

How should I handle and store recombinant Kallikrein-15 to maintain activity, and what is the shelf life?

Upon receipt, spin briefly, aliquot into single-use volumes, and store at -20°C. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides cryoprotection, but repeated freeze-thaw cycles will reduce serine protease activity through autolysis and aggregation. Shelf life is 12 months from the manufacturing date when stored correctly. For short-term use (within 1–2 weeks), working aliquots can be kept at 4°C. Do not dilute the stock in water alone — dilute into the assay buffer of choice immediately before use to maintain protein stability at lower concentrations.