Anti-Heparanase-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight bands should I expect for heparanase on a Western blot?

You should expect to see the 65 kDa latent pro-heparanase precursor in most cell lysates. If the enzyme has undergone proteolytic activation, you will detect two processed subunits at approximately 50 kDa and 8 kDa, forming the active heterodimer. The 8 kDa fragment may run off standard gels or appear faint due to its size. In highly invasive tumor lines or lysates enriched for secreted fractions, the processed forms predominate. Use reducing conditions and include a positive control lysate known to express heparanase, such as human placenta or certain carcinoma cell lines.

What dilution should I start with for Western blot using this heparanase antibody?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this rabbit polyclonal. Heparanase expression varies widely by cell type and activation state, so you may need to optimize between 1:500 and 1:2000 depending on your sample. Use standard blocking conditions with 5 percent non-fat milk or BSA in TBST. Incubate overnight at 4°C for best signal. If background is high, increase the dilution or switch blocking reagent. Always include a lysate control known to express heparanase to confirm antibody performance in your hands.

Does this heparanase antibody cross-react with mouse or rat samples?

This antibody is validated for human heparanase. Cross-reactivity with mouse or rat heparanase is not confirmed but may occur given the high sequence homology across mammalian orthologs, particularly in the catalytic domain. If you are working with rodent samples, we recommend testing the antibody empirically on positive control lysates from mouse or rat tissues known to express heparanase, such as placenta or certain tumor models. You may observe signal, but band intensity and specificity should be confirmed with appropriate knockout or knockdown controls to rule out non-specific binding.

Can I use this antibody for immunohistochemistry on paraffin-embedded tissue?

Yes, Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress. For heparanase IHC, antigen retrieval is typically required; citrate buffer at pH 6.0 with heat-induced epitope retrieval works well for most tissues. Heparanase localizes to lysosomes in resting cells but is secreted and accumulates in the extracellular matrix during tumor invasion or inflammation. Expect cytoplasmic and perinuclear staining in normal epithelium, with increased stromal and vascular staining in malignant or inflamed tissue. Optimize dilution empirically, starting around 1:100 to 1:200.

What positive control cell line or tissue should I use for heparanase expression?

Human placenta is an excellent positive control, as it expresses high levels of heparanase due to active trophoblast invasion and ECM remodeling. Among cell lines, several carcinoma lines including MCF-7 breast cancer, HepG2 hepatoma, and certain melanoma lines express detectable heparanase. Expression increases under hypoxic or inflammatory conditions, so consider treating cells with hypoxia or cytokines if baseline expression is low. For negative controls, heparanase knockout cell lines or siRNA knockdown samples are ideal to confirm antibody specificity and rule out off-target binding in your system.

How should I store this heparanase antibody and what is its shelf life?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is sufficient to prepare multiple single-use aliquots. Once thawed, the antibody remains stable at 4°C for up to one month if supplemented with 0.02 percent sodium azide or similar preservative. Avoid prolonged storage in frost-free freezers due to temperature cycling. Under proper storage conditions, rabbit polyclonal antibodies typically retain activity for two to three years from the manufacturing date. Check for precipitation or turbidity before use.

Why do I see multiple bands on my heparanase Western blot?

Multiple bands are common with heparanase due to its complex processing. The 65 kDa band represents the inactive precursor, while the 50 kDa and 8 kDa fragments indicate proteolytic activation. You may also observe intermediate processing forms or glycosylation variants, as heparanase contains N-linked glycosylation sites that shift its apparent molecular weight. Non-specific bands can arise if lysate protein is overloaded or if blocking is inadequate. To confirm specificity, include a heparanase knockdown or knockout control. If multiple genuine isoforms appear, it reflects the biological heterogeneity of heparanase maturation in your sample.

What lysis buffer and sample prep work best for detecting heparanase by Western blot?

Use a standard RIPA buffer or NP-40 lysis buffer supplemented with protease inhibitors to preserve heparanase integrity. Because heparanase is a lysosomal enzyme, include inhibitors for cysteine and aspartyl proteases such as E-64 and pepstatin A. Boil samples in reducing SDS-PAGE sample buffer to fully denature the protein. For detection of secreted heparanase, concentrate conditioned media by TCA precipitation or ultrafiltration before loading. Heparanase activity is pH-dependent and can be labile, so process samples quickly and store lysates at –80°C. Load 20 to 40 µg total protein per lane as a starting point.