Kallikrein-8 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Kallikrein-8 on SDS-PAGE and Western blot?

Recombinant KLK8 (UniProt O60259) has a calculated MW of approximately 28.5 kDa for the mature secreted sequence, but HEK293-derived protein typically migrates at 30–35 kDa under denaturing conditions due to N-linked glycosylation. On reducing SDS-PAGE you should see a single predominant band in that range at >90% purity. Under non-reducing conditions, the band position may shift slightly if disulfide-linked forms are present. Run a fresh preparation alongside a molecular weight ladder calibrated in the 20–50 kDa range for the cleanest result.

Is this recombinant Kallikrein-8 the zymogen or the active mature form?

This product is the active mature form — the propeptide has been removed during processing in HEK293 cells, and the catalytic triad (His-Asp-Ser) is intact and competent. It is not the zymogen/inactive proform. If your experiment requires controlled activation from a precursor, this is not the right format. For studies of KLK8 enzymatic activity against casein, fibrinogen, fibronectin, L1CAM, or fluorogenic peptide substrates, the active enzyme is ready to use directly from the aliquot after equilibration to assay temperature.

What substrates and fluorogenic peptides work best for a Kallikrein-8 activity assay?

KLK8 is a trypsin-like serine protease with a preference for Arg/Lys at the P1 position. The fluorogenic peptide Boc-Val-Pro-Arg-AMC is commonly used for in vitro activity assays and provides a clean linear readout. Native macromolecular substrates including casein, fibrinogen, and L1CAM have been validated biochemically. For gel-based readout, incubate with casein at 37°C for 30–60 min and visualize cleavage by SDS-PAGE. Assay buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl works well and is compatible with the supplied storage buffer — no desalting is typically required.

What assay buffer conditions and pH are optimal for recombinant Kallikrein-8 enzymatic activity?

KLK8 serine protease activity is optimal around pH 7.5–8.0. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is directly compatible with most activity assays at low enzyme dilutions, so glycerol carry-over is minimal. If you need to reduce glycerol below 0.1%, dialysis or gel filtration into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) is advisable. Including 0.01% BSA in the assay buffer helps prevent adsorptive losses at low protein concentrations (< 50 nM). Avoid reducing agents such as DTT above 1 mM, which can disrupt disulfide bonds essential for correct KLK8 folding.

What starting concentration of recombinant Kallikrein-8 should I use in an enzyme activity assay?

For fluorogenic peptide assays (e.g., Boc-Val-Pro-Arg-AMC at 100–200 µM substrate), a starting enzyme concentration of 1–10 nM is a reasonable range to establish linear kinetics. For native substrate cleavage (casein, fibrinogen), 50–200 nM KLK8 with a 30–60 minute incubation at 37°C typically yields visible cleavage bands. Titrate enzyme concentration in your first experiment to ensure you are in the linear portion of the velocity curve before running inhibitor IC50 measurements. All aliquots are supplied at a defined concentration stated on the CoA — use that as your stock reference.

Can I use recombinant Kallikrein-8 as a Western blot positive control for the RP-Kallikrein8 rabbit polyclonal antibody?

Yes — recombinant KLK8 (REC-Kallikrein8) is the recommended positive control for the matched RP-Kallikrein8 rabbit polyclonal antibody (see /anti-kallikrein-8-rabbit-polyclonal-antibody). Both are produced in-house and have been validated against the same antigen preparation. Load 20–50 ng of recombinant protein per lane on a 12% SDS-PAGE gel under reducing conditions. The antibody detects the recombinant and the endogenous protein at 30–35 kDa. This pairing is particularly useful for confirming antibody specificity alongside tissue lysates or conditioned medium samples where endogenous KLK8 expression may be low.

How much recombinant Kallikrein-8 should I load as a positive control to get a clean Western blot band?

20–50 ng per lane is the standard loading range when using the matched RP-Kallikrein8 antibody (SKU: RP-Kallikrein8) at a primary dilution of 1:1,000–1:2,000. At 20 ng you should see a clear band without significant background; at 50 ng the band is strong enough to serve as a reliable size marker reference at 30–35 kDa. If you are probing blots that also contain concentrated tissue lysate, load the recombinant in a flanking lane to avoid signal spillover. Running the positive control on every blot is especially valuable when working with low-expressing samples such as primary hippocampal neurons.

How should I store and handle recombinant Kallikrein-8 to preserve enzymatic activity over time?

Store at -20°C in single-use aliquots immediately upon receipt. Repeated freeze-thaw cycles progressively reduce enzymatic activity and should be avoided — aliquot to your typical experimental volume before the first use. Upon thawing, equilibrate on ice and use within the same working session where possible. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the protein at -20°C for at least 12 months from the manufacture date indicated on the CoA. Once thawed, keep on ice and complete assay setup within 2–4 hours. Do not dilute into water — use a compatible buffer to prevent denaturation at low ionic strength.