Heparanase 1 Clone HP3/17

What is Heparanase 1 and why is Clone HP3/17 significant for research?

Heparanase 1 (HPA1) is an endo-β-D-glucuronidase that specifically degrades heparan sulfate side chains of heparan sulfate proteoglycans (HSPGs) in the extracellular matrix. This enzymatic activity plays a crucial role in ECM degradation, which facilitates the migration and extravasation of tumor cells and inflammatory leukocytes. Additionally, heparanase releases growth factors and cytokines that stimulate cell proliferation and chemotaxis .

Clone HP3/17 is a highly specific mouse monoclonal antibody raised against a polypeptide from the 50 kDa subunit of heparanase. Its significance stems from its ability to recognize both the 65 kDa precursor and the 50 kDa active subunit of heparanase in human and mouse samples. This dual recognition capability makes it invaluable for tracking heparanase processing and activation in experimental systems .

What are the structural characteristics of Heparanase 1 relevant to Clone HP3/17 binding?

Heparanase exists as a heterodimer comprised of a 50 kDa subunit harboring the active site and an 8 kDa subunit. It is initially produced as a latent 65 kDa precursor that undergoes proteolytic processing to form its active heterodimeric structure. Clone HP3/17 was raised specifically against a polypeptide from the 50 kDa subunit of Heparanase, which contains the catalytic domain of the enzyme .

The antibody demonstrates high specificity with a purity of >98% on SDS-PAGE when loaded at 50 μg/lane. This specificity allows researchers to reliably detect both the precursor and processed forms of heparanase, making it useful for studying the enzyme's activation in various pathological conditions, especially cancer progression and metastasis .

What are the optimal storage and handling conditions for Clone HP3/17?

For optimal performance and longevity of Clone HP3/17, researchers should follow these storage and handling guidelines:

• Short-term storage: Store at 4°C. The antibody remains stable for up to six months from the date of shipment under these conditions .

• Long-term storage: For extended storage, freeze in working aliquots at -20°C .

• Avoid repeated freeze-thaw cycles as these can degrade antibody quality and diminish performance .

• The antibody is supplied as a 0.22 micron filtered solution of 20 mM Sodium Phosphate +150 mM NaCl, pH 7.2, containing 0.01% Thimerosal .

• Important precaution: Thimerosal is a poisonous and hazardous substance which should be handled by trained staff only with appropriate safety measures .

How should Clone HP3/17 be used in Western blotting protocols?

Clone HP3/17 can be effectively used in Western blotting following these methodological guidelines:

• Sample preparation:

  • Lyse cells in RIPA buffer (150 mM NaCl, 50 mM TRIS-HCl pH 8, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton-X) with Complete Protease Inhibitor Mixture .

  • Denature equal amounts of protein in reducing sample buffer for 10 minutes at 100°C .

• Electrophoresis and transfer:

  • Resolve protein samples in 10% SDS-PAGE gels .

  • Electrotransfer to nitrocellulose membranes .

• Blocking and antibody incubation:

  • Block non-specific binding with 5% non-fat milk in TBST buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) .

  • Incubate membranes with Clone HP3/17 antibody (MA1-83806, Thermo Fisher) overnight at 4°C .

  • Incubate with a secondary peroxidase-conjugated antibody for 1 hour at room temperature .

• Detection:

  • Detect signal using a chemiluminescent substrate like Luminata Forte Western HRP Substrate .

  • Acquire and document the signal using an appropriate imaging system .

The antibody should detect both the 65 kDa precursor and 50 kDa active subunit of heparanase in human and mouse samples.

What protocols should be followed for immunofluorescence studies with Clone HP3/17?

For immunofluorescence applications, the following protocol has been validated for Clone HP3/17:

• Cell fixation and permeabilization:

  • Fix cells in 4% paraformaldehyde for 15 minutes .

  • Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes .

  • Block with 3% bovine serum albumin (BSA) in PBS at room temperature for 30 minutes .

• Antibody incubation:

  • Incubate cells overnight at 4°C with Clone HP3/17 antibody diluted in PBS with 1% BSA .

  • Wash three times for 5 minutes each with PBS .

  • Incubate for 1 hour at 37°C with the appropriate secondary antibody diluted in PBS with 1% BSA .

• Nuclear staining and visualization:

  • Counterstain cell nuclei with Hoechst 33258 .

  • Obtain images using a confocal microscope (e.g., LeicaSP5) .

This method has been successfully used to visualize heparanase expression patterns in cancer cell lines, including DU145 and PC3 prostate cancer cells, allowing researchers to correlate heparanase localization with cellular phenotypes .

How can Clone HP3/17 be used to study heparanase's role in epithelial-mesenchymal transition (EMT)?

Clone HP3/17 has been instrumental in elucidating heparanase's role in EMT, particularly in prostate cancer. The following experimental approach demonstrates how the antibody can be used for such studies:

• Experimental model establishment:

  • Establish cell lines with modified heparanase expression (overexpression or silencing) using appropriate vectors .

  • For overexpression: Transfect cells with a plasmid coding HPSE ORF (e.g., DU145 cells) .

  • For silencing: Transfect cells with shRNAs targeting human heparanase (e.g., PC3 cells) .

  • Select stable transfectants using appropriate antibiotics (G418 for overexpression, puromycin for silencing) .

• Verification of heparanase expression:

  • Use Clone HP3/17 in Western blotting and immunofluorescence to confirm the efficacy of overexpression or silencing .

  • Compare with control cells transfected with empty vector or non-targeting shRNA .

• EMT marker analysis:

  • Assess expression of epithelial markers (E-cadherin) and mesenchymal markers (vimentin, α-SMA) via:

    • qRT-PCR using primers shown in Table 1 .

    • Western blotting with specific antibodies against E-cadherin, vimentin, and α-SMA .

    • Immunofluorescence for visualization of marker localization .

Research using this approach has demonstrated that heparanase acts as an EMT inducer in prostate cancer. Specifically, HPSE overexpression in DU145 cells reduced E-cadherin expression and increased mesenchymal markers, while HPSE silencing in PC3 cells had the opposite effect .

What methods can be used to quantify secreted heparanase levels in conjunction with Clone HP3/17?

For quantitative analysis of secreted heparanase levels, researchers can use ELISA in combination with Clone HP3/17 for validation:

• ELISA protocol:

  • Collect cell-conditioned media from experimental and control cells .

  • Use a commercial human heparanase ELISA kit (e.g., EIAab Science Ltd., Haifa, Israel) .

  • Follow the manufacturer's protocol, which typically employs a biotin-conjugated polyclonal antibody specific for heparanase .

  • Assess heparanase concentration by measuring color changes induced by the biotin-conjugated antibody and enzyme-conjugated avidin .

  • Compare the optical density (O.D.) of samples to a standard curve provided by the kit .

• Validation with Western blotting:

  • Process parallel samples for Western blotting using Clone HP3/17 .

  • Compare protein expression patterns with quantitative ELISA results.

  • This dual approach provides both quantitative (ELISA) and qualitative (Western blot) data on heparanase expression.

This combined methodology has been successfully applied in studies examining heparanase's role in drug resistance, such as lapatinib resistance in breast cancer , and could be adapted for various cancer models and experimental conditions.

How can researchers interpret the relationship between heparanase and syndecan-1 using Clone HP3/17?

Studies using Clone HP3/17 have revealed a complex relationship between heparanase and syndecan-1, particularly in the context of EMT and cancer progression:

• Experimental approach:

  • Establish cell models with modified heparanase expression (overexpression/silencing) .

  • Confirm heparanase status using Clone HP3/17 in Western blotting and immunofluorescence .

  • Analyze syndecan-1 expression at the gene level using qRT-PCR (see Table 1 for primers) .

  • Quantify secreted syndecan-1 levels using ELISA (e.g., human syndecan ELISA kit from Abcam) .

• Data interpretation:

  • Research has demonstrated that heparanase expression inversely correlates with syndecan-1 expression .

  • In DU145 cells, HPSE overexpression reduced syndecan-1 levels .

  • In PC3 cells, HPSE silencing increased syndecan-1 expression .

  • This inverse relationship supports the model where TGF-β induces EMT by activating SNAI-1, which represses syndecan-1 expression .

  • The coordinated loss of syndecan-1 and E-cadherin has been documented in many epithelial malignancies .

• Biological significance:

  • Syndecan-1 is necessary for maintaining the epithelial phenotype .

  • Its depletion during malignant transformation alters cell morphology and anchorage-dependent growth .

  • In prostate cancer, changes in syndecan-1 expression are linked to EMT .

  • Syndecan-1 expression is lower in PC3 and DU145 prostate cancer cell lines than in normal prostate epithelial cells .

These findings indicate that researchers can use Clone HP3/17 as part of a multi-marker approach to study the heparanase-syndecan-1 axis in cancer progression, providing insights into EMT regulation and potential therapeutic targets.

What are the potential mechanisms of heparanase involvement in cancer stem cell properties that can be studied using Clone HP3/17?

Clone HP3/17 has been instrumental in revealing heparanase's role in regulating cancer stem cell (CSC) properties, particularly in prostate cancer:

• Experimental approach:

  • Establish cell models with modified heparanase expression (overexpression/silencing) .

  • Confirm heparanase expression using Clone HP3/17 in Western blotting and immunofluorescence .

  • Analyze stemness markers at the gene level using qRT-PCR with specific primers :

• Additional functional assays:

  • Colony formation assay: Seed 1,000 cells per well in 35-mm culture dishes and incubate for 7-10 days. Fix with paraformaldehyde, stain with 0.1% crystal violet, and count colonies (defined as >50 cells) .

  • This assay assesses the self-renewal and proliferative capacity of cells, which are key properties of cancer stem cells.

• Data interpretation and biological significance:

  • Research has shown that heparanase modulates the expression of stemness markers SOX2, OCT4, and NANOG in prostate cancer cells .

  • These transcription factors are crucial for maintaining pluripotency and self-renewal in stem cells.

  • The link between heparanase, EMT, and cancer stem cell properties suggests that heparanase might be a potential therapeutic target for preventing cancer metastasis and recurrence.

By combining Clone HP3/17-based detection of heparanase with analyses of stemness markers and functional assays, researchers can gain deeper insights into the mechanisms by which heparanase contributes to cancer stem cell phenotypes and tumor aggression.

What are common issues encountered when using Clone HP3/17 and how can they be addressed?

When working with Clone HP3/17, researchers might encounter several technical challenges. Here are common issues and their solutions:

• Weak or no signal in Western blotting:

  • Ensure proper sample preparation with complete lysis using RIPA buffer with protease inhibitors .

  • Verify protein concentration using a reliable method (BCA or Bradford assay).

  • Confirm transfer efficiency using a reversible protein stain before blocking.

  • Consider extending primary antibody incubation to overnight at 4°C .

  • Increase antibody concentration if necessary, but maintain specificity.

  • Use a sensitive detection reagent like Luminata Forte Western HRP Substrate .

• High background in immunofluorescence:

  • Ensure thorough blocking with 3% BSA in PBS .

  • Increase washing steps (3 times for 5 minutes each) after antibody incubations .

  • Dilute primary and secondary antibodies appropriately in 1% BSA/PBS .

  • Include negative controls (omitting primary antibody) to assess non-specific binding.

• Inconsistent results across experiments:

  • Avoid repeated freeze-thaw cycles of antibody, as this can reduce activity .

  • Prepare working aliquots for long-term storage at -20°C .

  • Standardize cell culture conditions and sample preparation protocols.

  • Include appropriate positive controls (cell lines with known heparanase expression).

• Difficulty distinguishing between 65 kDa precursor and 50 kDa active forms:

  • Use higher resolution SDS-PAGE (12% instead of 10%) to better separate these closely sized proteins.

  • Include positive controls with known processing status of heparanase.

  • Consider using reducing conditions to fully denature protein samples .

Addressing these issues through careful optimization of protocols will ensure reliable and reproducible results when using Clone HP3/17 for heparanase detection and characterization.