Kallikrein-2 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Kallikrein-2 on SDS-PAGE or Western blot?

The mature recombinant KLK2 runs at approximately 28–30 kDa under reducing SDS-PAGE conditions, consistent with the processed form after signal-peptide cleavage from the 261-amino-acid precursor (UniProt P20151). HEK293 expression supports native N-glycosylation, so the observed band may migrate slightly higher than the calculated ~28 kDa depending on glycan load. Under non-reducing conditions, disulfide-stabilized dimers can appear near 55–60 kDa. Purity is >95% by SDS-PAGE; the dominant Coomassie band should be unambiguous at this position.

What processing form of KLK2 is this recombinant — pro-form or mature active enzyme?

This preparation is the mature, catalytically active form of KLK2, with the signal peptide and propeptide removed during HEK293-mediated secretion and processing. The trypsin-like catalytic domain is intact, and the Ser-His-Asp active-site triad is preserved. You do not need to activate this material with an exogenous protease before use. If your experiment specifically requires the zymogen (pro-KLK2) for activation studies, this product is not appropriate — the mature active enzyme is what is supplied.

What substrates does Kallikrein-2 cleave, and what fluorogenic substrate works best for activity assays?

KLK2 is a serine protease that preferentially cleaves Met-Lys and Arg-Ser bonds, releasing Lys-bradykinin from kininogen in vivo. For in vitro activity assays, the fluorogenic substrate H-D-Val-Leu-Arg-AMC (VLR-AMC) is well-characterized for KLK2 and gives a robust signal. Boc-Phe-Ser-Arg-AMC is an alternative with good selectivity. Chromogenic substrates such as H-D-Pro-Phe-Arg-pNA are also compatible. A starting enzyme concentration of 0.5–2 nM in assay buffer with 100–200 µM substrate is a reasonable starting point for Km determinations.

What assay buffer should I use for Kallikrein-2 activity assays, and is the storage buffer compatible?

For fluorogenic or chromogenic cleavage assays, use 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20 as a starting condition — KLK2 shows good activity in this range. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with direct dilution into assay buffer; glycerol carry-over at typical working dilutions (≤1%) does not measurably inhibit activity. Avoid EDTA above 1 mM in the assay; KLK2 is a serine protease but trace metal chelation can affect substrate stability in some AMC-based systems.

What starting concentration of recombinant KLK2 is recommended for small-molecule inhibitor IC50 experiments?

For IC50 determinations, 1–5 nM recombinant KLK2 is a practical working range, keeping enzyme well below substrate Km to satisfy Morrison tight-binding or standard Cheng-Prusoff correction assumptions. Titrate your inhibitor across at least 8 concentrations spanning 3 log units bracketing the anticipated Ki. Run each inhibitor concentration against 100 µM VLR-AMC substrate in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20. Include a vehicle control (≤1% DMSO) to confirm solvent tolerance before full dose-response.

Can I use recombinant Kallikrein-2 as a positive control for Western blot with the matched rabbit polyclonal antibody?

Yes — this is one of the primary intended use cases. The matched antibody (SKU: RP-Kallikrein2) is validated for Western blot and was raised against KLK2, making it directly compatible with this recombinant as a positive control. Load 50–200 ng of recombinant KLK2 per lane on a 12% or 4–20% gradient gel; expect a clean band at ~28–30 kDa. This pairing is particularly useful for antibody validation experiments where a defined, structurally intact antigen is required alongside cell lysate lanes.

How much recombinant Kallikrein-2 should I load per lane to get a clean positive-control band on Western blot?

Load 50–200 ng per lane for a reliable signal using the matched RP-Kallikrein2 rabbit polyclonal antibody. At 50 ng, the band at ~28–30 kDa is detectable with standard HRP-conjugated secondary antibodies (1:5,000–1:10,000) and chemiluminescent detection. At 200 ng you get a strong reference band suitable for quantitative comparisons or if your detection system is less sensitive. Run the recombinant alongside cell or tissue lysates to confirm antibody specificity in your experimental system — see SKU RP-Kallikrein2 (/anti-kallikrein-2-rabbit-polyclonal-antibody) for validated dilution ranges.

How should I handle, dilute, and store recombinant Kallikrein-2 to preserve activity over time?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which progressively reduce serine protease activity. On the day of use, thaw on ice and dilute into your assay or storage buffer immediately before the experiment — do not leave the diluted enzyme at room temperature for extended periods. Working aliquots can be held at 4°C for up to 48 hours without significant activity loss. Endotoxin is <0.1 EU/µg by LAL, suitable for cell-based assays.