Heparanase-1 (Recombinant)

Frequently Asked Questions

What molecular weight bands should I expect from recombinant Heparanase-1 on SDS-PAGE or Western blot?

REC-Heparanase1 is expressed as the full-length 543 aa precursor in HEK293 cells and undergoes proteolytic processing into an 8 kDa and a 50 kDa subunit — the two forms that associate non-covalently to constitute the active heterodimer. Under reducing SDS-PAGE conditions you should see a dominant band at ~50 kDa and, depending on loading amount and staining sensitivity, a secondary band near 8 kDa. The full-length precursor (~65 kDa with glycosylation) may also appear as a minor species. Purity is >90% by SDS-PAGE, so these are the bands you should budget for when interpreting blots.

What is the active form of recombinant Heparanase-1 and how does the proteolytic processing work?

The native human enzyme (UniProt Q9Y251) is translated as a 543 aa inactive precursor that is cleaved to remove a linker segment, yielding an 8 kDa N-terminal subunit and a 50 kDa C-terminal subunit. These two subunits re-associate as a non-covalent heterodimer, which is the catalytically active form. REC-Heparanase1 is produced in HEK293 cells and supplied in the processed, active heterodimeric state. Because HEK293 cells recapitulate mammalian glycosylation, the glycoform profile closely mirrors native HPSE — a relevant advantage when glycoform-dependent substrate recognition or inhibitor binding is part of your experimental rationale.

What substrate does recombinant Heparanase-1 cleave and at what cleavage site?

Heparanase-1 is a mammalian endo-β-glucuronidase that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs) at specific glucuronic acid–N-sulfo glucosamine linkages. For in vitro activity assays, biotinylated or fluorescently labeled heparan sulfate substrates (e.g., HS oligosaccharide-based FRET substrates) are most commonly used. Standard solid-phase HS degradation assays using 35S-labeled HSPGs or commercially available fluorescent HS substrates at 0.5–5 µg/reaction also work well. Activity is pH-dependent — assays run optimally in the pH 5.0–6.0 range, consistent with the enzyme's lysosomal origin.

What buffer and pH conditions should I use for a Heparanase-1 activity assay in vitro?

REC-Heparanase1 has a pronounced pH optimum in the acidic range, typically pH 5.0–6.0, consistent with its lysosomal localisation. For activity assays, a standard buffer is 50 mM sodium acetate or MES, pH 5.4, supplemented with 1–5 mM DTT to maintain reducing conditions. The storage buffer for this recombinant is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — dilute the enzyme into your assay buffer immediately before use. Avoid extended incubation at neutral or alkaline pH, as enzymatic activity drops significantly outside the pH 4.5–6.5 window.

What starting concentration of recombinant Heparanase-1 should I use for an IC50 inhibitor profiling assay?

For IC50 determinations, begin with 0.1–1 µg/mL of REC-Heparanase1 in your assay buffer (pH 5.4). This concentration range typically gives a robust, linear signal with fluorescent HS substrates at 10–50 µg/mL substrate concentration, while remaining within the dynamic range needed to detect inhibition across a broad compound concentration series. Run a substrate saturation curve first to confirm you are not substrate-limited. Because mammalian glycoforms can influence inhibitor binding affinity relative to bacterially expressed enzyme, the HEK293-derived protein here is the preferred choice for inhibitor profiling intended to translate to cellular or in vivo settings.

Can I use REC-Heparanase1 as a positive control for Western blot with the matched anti-Heparanase-1 antibody?

Yes — this is one of the most straightforward use cases. REC-Heparanase1 is specifically paired with RP-Heparanase1, the rabbit polyclonal antibody available at /anti-heparanase-1-rabbit-polyclonal-antibody. The antibody has been validated for Western blot detection of the processed 50 kDa subunit, which is the dominant species in this recombinant preparation. Load 20–50 ng of REC-Heparanase1 per lane alongside your cell or tissue lysates; this quantity reliably produces a clear 50 kDa signal without saturating the band. Using the matched pair also confirms antibody lot-to-lot consistency — a practical advantage for long-running studies.

How much recombinant Heparanase-1 should I load for a Western blot positive control lane?

For a standard chemiluminescent Western blot using RP-Heparanase1, load 20–50 ng of REC-Heparanase1 per lane. At 20 ng you should see a clean ~50 kDa band with most standard HRP-conjugated secondary antibodies at 1:5,000–1:10,000 dilution. If you are running a less sensitive detection method, increase to 50 ng. Avoid loading more than 100 ng — above this threshold the band can smear or bleed into adjacent sample lanes. The 8 kDa subunit may require higher loading (100–200 ng) and a higher-percentage gel (15–18%) for reliable detection.

How should I store and handle REC-Heparanase1 to maintain enzymatic activity long-term?

REC-Heparanase1 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. Repeated freeze-thaw cycles cause measurable loss of specific activity — aliquot upon first receipt if the lot was shipped as a single vial. For short-term working use (1–3 days), the enzyme is stable at 4°C. When diluting for assay, use a carrier protein such as 0.1% BSA in your assay buffer to reduce adsorptive losses at low protein concentrations. Endotoxin is <0.1 EU/µg by LAL assay, making this preparation compatible with cell-based and primary culture experiments.