Heparanase-2 (Recombinant)

Frequently Asked Questions

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

The HPSE2 polypeptide backbone is 592 amino acids, giving a calculated MW of approximately 65 kDa. However, HPSE2 is a secreted glycoprotein produced here in HEK293 cells, so native-like N-glycosylation pushes the apparent MW on SDS-PAGE to roughly 72–80 kDa under denaturing, reducing conditions. Run alongside a broad-range ladder and expect a diffuse or slightly smeared band — characteristic of glycoproteins. If you see a sharper band near 65 kDa, partial deglycosylation during sample prep is the likely cause. PNGase F treatment will collapse the band to ~65 kDa as a useful control.

Does recombinant HPSE2 have heparanase enzymatic activity or is it catalytically inactive?

HPSE2 is catalytically inactive. Unlike HPSE1, it lacks the two glutamate residues (E225 and E343 in HPSE1) that constitute the endo-β-glucuronidase active site. This recombinant will not cleave heparan sulfate chains. Its documented biological function is inhibitory: HPSE2 binds heparin and heparan sulfate proteoglycans with high affinity and competes with HPSE1 for substrate access, suppressing HPSE1-mediated HS degradation. If you are designing an HPSE1 inhibition assay, HPSE2 serves as the inhibitory protein, not the enzyme. Use active recombinant HPSE1 as the enzymatic component.

How do I set up an HPSE1 inhibition assay using recombinant Heparanase-2?

Use a fluorometric heparan sulfate degradation assay with HPSE1 as the enzyme and BODIPY- or FITC-labeled heparan sulfate as substrate. Pre-incubate recombinant HPSE2 with the HS substrate for 15–30 min at room temperature before adding HPSE1 to allow competitive binding. HPSE1 assay buffer (50 mM sodium acetate pH 5.0, 150 mM NaCl) is compatible with this recombinant. Titrate HPSE2 from 10 nM to 1 µM to establish the inhibitory curve. Published IC50 values for HPSE2 inhibition of HPSE1 fall in the 50–200 nM range depending on substrate concentration and assay format.

What buffer is recombinant Heparanase-2 supplied in and can I dilute it directly into my assay?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For HPSE1 inhibition assays run at pH 5.0 (sodium acetate buffer), dilute HPSE2 at least 1:5–1:10 into your assay buffer immediately before use so the Tris and glycerol contribution is negligible. Avoid repeated freeze-thaw; working aliquots can be held at 4°C for up to 48 hours. Add 0.1% BSA as a carrier if diluting below 10 nM to prevent adsorptive losses on tube surfaces. Do not sonicate or vortex — mix by gentle pipetting.

What starting concentration of recombinant Heparanase-2 is recommended for heparin binding or SPR binding experiments?

For surface plasmon resonance or biolayer interferometry with heparin-coated chips, start at 100–500 nM HPSE2 in running buffer (PBS or 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20). KD values for HPSE2–heparin interactions reported in the literature are in the low nanomolar to sub-nanomolar range, so use a concentration series spanning 1–500 nM for full kinetic fitting. For pull-down or co-precipitation with heparan sulfate proteoglycans in cell lysates, 200–500 nM recombinant HPSE2 per reaction is a reasonable starting point. Confirm protein integrity by SDS-PAGE before each experiment.

Can I use recombinant Heparanase-2 as a positive control for Western blot with the matched anti-heparanase-2 antibody?

Yes — this is one of the primary intended uses. The matched rabbit polyclonal antibody (SKU: RP-Heparanase2; see /anti-heparanase-2-rabbit-polyclonal-antibody) was raised and validated in the same laboratory that produced this recombinant, so compatibility is confirmed rather than assumed. Load 20–50 ng of recombinant HPSE2 per lane on a 10% SDS-PAGE gel. Expect a band at ~72–80 kDa. This lane gives you a clean, reproducible reference band alongside your cell lysate or tissue samples, which is particularly useful when endogenous HPSE2 expression is low.

How much recombinant Heparanase-2 should I load for Western blot positive control and what antibody dilution should I use?

Load 20–50 ng of recombinant HPSE2 per lane. At 50 ng, the band at ~72–80 kDa is clearly detectable; dropping to 20 ng still gives a usable signal and keeps the positive control lane from overwhelming your chemiluminescent exposure. For the matched antibody RP-Heparanase2, a primary dilution of 1:500–1:2000 in 5% non-fat milk/TBST is a reasonable starting range — refer to the antibody datasheet for lot-specific recommendations. Use standard HRP-conjugated anti-rabbit secondary at 1:5000–1:10,000. Purity of this recombinant is >95% by SDS-PAGE, so no contaminating bands should confound interpretation.

What are the storage and shelf life conditions for recombinant Heparanase-2 and how do I handle aliquots?

Store at -20°C in single-use aliquots immediately upon receipt. Under these conditions, the protein is stable for at least 12 months. Once thawed, keep on ice and use within 24–48 hours; do not refreeze. Repeated freeze-thaw cycles cause aggregation and activity loss for most glycoproteins — HPSE2 is no exception. Aliquot size should match your typical experiment: for inhibition assays consuming 200–500 nM per well, a 10 µg aliquot covers multiple runs. Purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL assay, so the preparation is suitable for cell-based experiments without additional endotoxin removal.