Kallikrein-3 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Kallikrein-3 on SDS-PAGE or Western blot?

The mature, enzymatically active form of Kallikrein-3 (after removal of the 17-residue signal peptide and 7-residue propeptide) has a predicted polypeptide MW of approximately 26 kDa. Because this recombinant is produced in HEK293 cells with native-like N-linked glycosylation, the apparent MW on reducing SDS-PAGE typically runs at 28–33 kDa — a diffuse band is normal and reflects heterogeneous glycosylation. Under non-reducing conditions, the band may shift slightly due to intramolecular disulfide constraints. If you observe a doublet, this is consistent with partially processed pro-forms co-purifying with the mature enzyme.

Is this recombinant PSA the proenzyme form or the mature enzymatically active form of Kallikrein-3?

This product is supplied as the mature, active form of Kallikrein-3 (EC 3.4.21.77), corresponding to residues 25–261 of UniProt P07288 after cleavage of both the signal peptide (residues 1–17) and the activation propeptide (residues 18–24). It is not the zymogen (pro-PSA). Enzymatic activity is confirmed prior to lot release. If your experiment specifically requires the proPSA isoform — relevant in certain cancer biomarker contexts — note that this product will not model the uncleaved precursor state.

What substrates does recombinant Kallikrein-3 cleave, and what synthetic substrate can I use in an activity assay?

Kallikrein-3 is a chymotrypsin-like serine endopeptidase that cleaves semenogelin-1 and semenogelin-2 at specific Tyr and Leu residues in its physiological context. For in vitro activity assays, the fluorogenic substrate H-Ser-Ser-Lys-Leu-Gln-AMC (or the commercially available Boc-Gln-Ala-Arg-AMC as a secondary option) is commonly used and well-documented in the literature. Cleavage is measured by AMC release at excitation 360 nm / emission 460 nm. Confirm activity is zinc-independent (Kallikrein-3 is a serine protease, not a metalloprotease), so avoid including EDTA in your assay buffer above 0.1 mM.

What buffer conditions should I use for a Kallikrein-3 enzymatic activity assay, and what is the recommended starting enzyme concentration?

For activity assays, a working buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl with 0.01% Tween-20 to prevent surface adsorption is a practical starting point — this is compatible with the storage buffer this protein ships in. A starting enzyme concentration of 1–5 nM is appropriate for fluorogenic substrate assays at substrate concentrations spanning the expected Km (typically 50–500 µM for AMC-conjugated peptides). Pre-incubate the enzyme at 37°C for 10 minutes before initiating the reaction. For IC50 determination of serine protease inhibitors, titrate enzyme at 2 nM to maintain linear substrate turnover throughout the inhibitor concentration range.

Can I use recombinant Kallikrein-3 as a positive control for Western blot with the matched rabbit polyclonal antibody RP-Kallikrein3?

Yes — this is a validated application. The matched rabbit polyclonal antibody (SKU: RP-Kallikrein3, available at /anti-kallikrein-3-rabbit-polyclonal-antibody) is produced and validated in the same laboratory against the mature Kallikrein-3 protein, making REC-Kallikrein3 the natural positive control for any Western blot run with RP-Kallikrein3. Both the antigen used for immunization and this recombinant are HEK293-expressed, so epitope conformation is matched. Expect a band at 28–33 kDa (glycosylated) with no ambiguity about which band is Kallikrein-3 when loaded alongside your cell lysate or tissue samples.

How much recombinant Kallikrein-3 should I load per lane as a Western blot positive control?

Load 50–200 ng of REC-Kallikrein3 per lane for standard Western blot with RP-Kallikrein3 antibody. At 50 ng, a clean single band at 28–33 kDa is detectable with chemiluminescent detection (ECL) at typical antibody dilutions (1:1,000–1:5,000). If your secondary antibody or detection system is less sensitive, scale up to 100–200 ng. Avoid loading more than 500 ng, as overloading a glycoprotein of this MW range tends to produce smearing that obscures the band relative to your lysate lanes. Run in a dedicated lane adjacent to your experimental samples rather than mixing into a single lane.

How should I handle, dilute, and store recombinant Kallikrein-3 to preserve enzymatic activity over time?

REC-Kallikrein3 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and stored at −20°C in single-use aliquots. Purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL assay. Avoid repeated freeze-thaw cycles — activity loss per cycle is measurable for serine proteases of this class. For working dilutions, use the storage buffer or your assay buffer supplemented with 0.1% BSA as a carrier to prevent adsorption at low concentrations (<10 nM). Diluted enzyme should be prepared fresh on the day of the experiment and kept on ice. Do not store working dilutions at 4°C for more than 24 hours.

Does recombinant Kallikrein-3 from HEK293 cells have full enzymatic activity compared to E. coli-expressed PSA?

HEK293 expression is specifically preferred for Kallikrein-3 because the protein requires correct disulfide bond formation across its three conserved disulfide bridges and N-linked glycosylation for full enzymatic activity and proper antigenic presentation — neither of which is reliably achieved in bacterial systems without refolding. E. coli-expressed Kallikrein-3 typically requires denaturation and refolding steps that result in variable specific activity and altered glycan profiles. This recombinant is assayed for chymotrypsin-like serine protease activity prior to lot release, and the specific activity is reported on the lot-specific certificate of analysis provided with each shipment.