Plasma Kallikrein (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Plasma Kallikrein run at on SDS-PAGE under reducing vs. non-reducing conditions?

Under reducing SDS-PAGE conditions, this HEK293-expressed recombinant resolves as two bands: a heavy chain of approximately 52–56 kDa and a catalytic light chain of approximately 33–36 kDa, reflecting the disulfide-linked two-chain active form. Under non-reducing conditions, the disulfide bond is preserved and you should observe a single band near 88–92 kDa. N-linked glycosylation from the mammalian expression system adds heterogeneous mass, so bands may appear slightly diffuse or run above predicted sequence mass (~71 kDa unprocessed). Purity is >90% by SDS-PAGE.

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

This product is supplied as the active, two-chain form — not the single-chain zymogen prekallikrein. The heavy chain (containing the apple domains responsible for HMWK binding) and the catalytic light chain (bearing the trypsin-like serine protease triad) are connected by a single disulfide bond. No additional activation step is required before use. If your experiment specifically requires the zymogen form (single-chain, ~85 kDa), contact us — but for most proteolytic or kinin-generation assays, the active enzyme is the correct starting material.

What substrates does recombinant Plasma Kallikrein cleave and what chromogenic or fluorogenic substrate should I use for activity assays?

Plasma kallikrein (EC 3.4.21.34) cleaves high-molecular-weight kininogen (HMWK) to release bradykinin and reciprocally activates factor XII. For routine activity assays, the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) is the established benchmark — monitor absorbance at 405 nm. A fluorogenic alternative is Boc-Val-Pro-Arg-AMC, read at Ex/Em 380/460 nm. Both substrates are cleaved after the Arg residue consistent with the enzyme's trypsin-like specificity. Run reactions in 50 mM Tris-HCl pH 7.8, 100–150 mM NaCl, 0.01% Tween-20 at 37°C for reliable initial-rate kinetics.

What buffer conditions are optimal for Plasma Kallikrein activity assays and is the storage buffer compatible?

Optimal activity is observed in 50 mM Tris-HCl pH 7.5–8.0, 100–150 mM NaCl, 0.01% Tween-20 at 37°C. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is close to optimal, so diluting the enzyme directly into assay buffer introduces minimal glycerol carry-over at working concentrations. Avoid reducing agents such as DTT or β-mercaptoethanol — these disrupt the disulfide bond linking the heavy and light chains and will abolish activity. EDTA at standard chelation concentrations is tolerated; this is not a metalloproteinase.

What starting concentration of recombinant Plasma Kallikrein should I use for a contact pathway activation or kinin-generation assay?

For HMWK cleavage and bradykinin-release assays, a useful starting point is 1–10 nM recombinant plasma kallikrein in your reaction volume. For factor XII activation feedback loop studies, titrate from 0.5–5 nM to avoid saturating the reciprocal activation system before steady state. For IC50 determination against a kallikrein inhibitor (e.g., aprotinin, C1-inhibitor), 2–5 nM enzyme with the S-2302 chromogenic substrate at 0.5 mM gives a robust signal window. Pre-warm enzyme to 37°C and initiate reactions by substrate addition; include a no-enzyme blank for background correction.

Can I use recombinant Plasma Kallikrein as a positive control for Western blot with the matched anti-Plasma Kallikrein antibody (RP-Plasma Kallikrein)?

Yes — this is one of the primary intended uses of this recombinant alongside SKU RP-Plasma Kallikrein (/anti-plasma-kallikrein-1b-rabbit-polyclonal-antibody). Because both are produced in the same laboratory, lot-to-lot compatibility is guaranteed. Load 20–50 ng per lane under reducing conditions; the antibody detects both the heavy chain (~52–56 kDa) and the light chain (~33–36 kDa), giving you a two-band positive control that confirms the antibody is resolving the processed form correctly. This pairing is particularly useful when validating RP-Plasma Kallikrein for plasma or liver lysate Western blots.

How much recombinant Plasma Kallikrein should I load for Western blot positive control and what band pattern should I expect?

Load 20–50 ng under reducing conditions for a clean, readily detectable signal with RP-Plasma Kallikrein at 1:1,000–1:2,000 dilution. Expect two bands: the heavy chain at ~52–56 kDa and the catalytic light chain at ~33–36 kDa. Both bands may appear slightly smeared due to heterogeneous N-glycosylation from the HEK293 expression system — this is normal and mirrors what you would see in human plasma. If you wish to visualize the intact disulfide-linked form (~88–92 kDa), run the sample under non-reducing conditions and omit β-mercaptoethanol from sample buffer.

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

Aliquots are stored at -20°C in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Upon first use, thaw on ice and dilute into ice-cold assay buffer; avoid vortexing — mix by gentle pipetting. Carrier protein (0.1% BSA) is recommended when diluting below ~10 µg/mL to prevent surface adsorption losses. Do not refreeze working dilutions; discard unused diluted material. Intact aliquots are stable for ≥12 months at -20°C. A single freeze-thaw cycle of the stock is acceptable, but activity loss can exceed 20% with repeated cycles, so single-use aliquot format is strongly advised.