Kallikrein-7 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-7 on SDS-PAGE and Western blot?

The KLK7 precursor (253 aa; UniProt P49862) has a predicted MW of ~28 kDa, but the HEK293-expressed recombinant runs at approximately 30–32 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation at the mature secreted form. Signal peptide cleavage is confirmed during processing in HEK293 cells, so you should not see the full-length unprocessed form. Under non-reducing conditions, intra-chain disulfide bonds can cause slight upward mobility shifts. Purity is >95% by SDS-PAGE, so the dominant band should be clean at that position.

What processing form of Kallikrein-7 is present in this recombinant — zymogen or active enzyme?

This preparation is the active, mature enzyme rather than the inactive zymogen. HEK293 expression supports the signal peptide cleavage and propeptide processing required to generate the catalytically competent form. The activation peptide (residues preceding the mature chain) is removed during secretion and downstream purification steps. If your experiment requires controlled zymogen-to-active conversion kinetics — for example, studying KLK cascade activation — you would need to generate the propeptide form separately; this product is not suitable for that specific application.

What substrates can I use to measure Kallikrein-7 chymotryptic activity in a fluorescence assay?

KLK7 is a chymotryptic serine protease (EC 3.4.21.117) that cleaves C-terminal to aromatic residues (Tyr, Phe at P1). The most commonly used fluorogenic substrate is Suc-Ala-Ala-Pro-Phe-AMC (or Phe-pNA for colorimetric readout). For a well-characterized peptide substrate, the insulin A-chain cleavage site at Tyr14–Gln15 can be used in HPLC-based assays. Assay buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl matches the storage buffer, minimizing dilution artifacts. Run a substrate titration from 50–500 µM AMC-peptide to establish Km before committing to inhibitor IC50 studies.

What buffer conditions and pH are optimal for recombinant Kallikrein-7 activity assays?

KLK7 chymotryptic activity is optimal between pH 7.0–8.5. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with direct use in functional assays; dilute the glycerol to ≤1% final in your assay volume to avoid enzyme stabilization artifacts. Including 0.01% BSA or 0.05% Tween-20 in the assay buffer helps prevent surface adsorption at low protein concentrations. Avoid PMSF, DFP, or other serine protease inhibitors in the assay buffer; they will irreversibly inhibit KLK7 activity.

What starting concentration of recombinant Kallikrein-7 should I use for an inhibitor IC50 assay?

For AMC-based fluorogenic assays, a starting enzyme concentration of 5–20 nM is typical, adjusted so that substrate turnover is linear (<10% conversion) over the measurement window. At concentrations below 2 nM, surface adsorption becomes significant — include carrier protein (0.01% BSA) if you need to go that low. For IC50 determination, keep enzyme concentration well below the Ki of your inhibitor of interest; tight-binding inhibitors will require Morrison equation fitting if enzyme and inhibitor concentrations are comparable. Titrate enzyme activity empirically on your plate reader setup before committing to a full inhibitor screen.

Can I use recombinant Kallikrein-7 as a positive control for Western blot with the matched TPB antibody RP-Kallikrein7?

Yes — this is a primary validated use case for this product paired with RP-Kallikrein7 (/anti-kallikrein-7-rabbit-polyclonal-antibody). Load 10–50 ng per lane under reducing conditions; at 50 ng the band at ~30–32 kDa is consistently detectable with RP-Kallikrein7 at a 1:1000–1:2000 dilution using standard HRP-conjugated secondary antibodies. Because both products come from the same lab and have been cross-validated, this pairing is reliable for antibody lot-to-lot QC and for verifying antibody performance in new sample matrices before running experimental lysates.

How much recombinant Kallikrein-7 should I load per lane for a Western blot positive control?

Load 25–50 ng per lane for a robust positive control band. At 25 ng you will typically see a clean band at ~30–32 kDa with RP-Kallikrein7 (SKU: RP-Kallikrein7) at 1:1000 dilution after 60-minute HRP incubation and standard ECL. If you are using the recombinant to spike into a complex lysate background for antibody validation, 10–25 ng spiked into 20–30 µg total lysate protein is usually sufficient for the recombinant band to be distinguishable. Titrate a 2-fold dilution series (100, 50, 25, 12.5 ng) on your first blot to establish your lab's specific detection threshold.

How should I handle, dilute, and store recombinant Kallikrein-7 to preserve activity over time?

The protein is shipped in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C in single-use aliquots immediately upon receipt. Repeated freeze-thaw cycles cause significant activity loss — aliquot to your typical single-experiment volume before freezing. For working dilutions, use the storage buffer or your assay buffer supplemented with 0.01% BSA; prepare fresh dilutions on ice and use within 4 hours. Avoid diluting below ~50 µg/mL without carrier protein present. Endotoxin is <0.1 EU/µg by LAL, so the preparation is suitable for cell-based assays where LPS contamination is a concern.