HAS3 Antibody

What is HAS3 and why is it important for research?

HAS3 (hyaluronan synthase 3) is one of three isoenzymes responsible for cellular hyaluronan synthesis. It catalyzes the addition of GlcNAc or GlcUA monosaccharides to nascent hyaluronan polymers, which are major components of extracellular matrices. HAS3 is essential for hyaluronan synthesis and plays critical roles in tissue architecture regulation, cell adhesion, migration, and differentiation . Unlike other hyaluronan synthases (HAS1 and HAS2), HAS3 produces lower molecular weight hyaluronan and is considered the most active isoform in adults . Research on HAS3 is particularly valuable for understanding extracellular matrix dynamics and cancer biology, as HAS3 inhibition has been shown to decrease tumor growth in certain cancers .

What are the primary research applications for HAS3 antibodies?

HAS3 antibodies are primarily used in multiple molecular and cellular techniques:

These applications facilitate both expression analysis and functional studies of HAS3 in various tissue and cellular contexts .

How can I confirm the specificity of an HAS3 antibody?

Confirming antibody specificity for HAS3 requires multiple validation approaches:

• Positive control testing: Use tissues/cells known to express HAS3 (e.g., human fetal lung, kidney, uterus lysates)

• siRNA knockdown: Compare antibody reactivity in wild-type versus HAS3-silenced samples

• Western blot analysis: Verify the predicted 63 kDa band size

• Cross-reactivity assessment: Test against related proteins (HAS1, HAS2) to ensure specificity

• Immunocytochemistry: Compare staining patterns with published data on HAS3 localization

A combination of these methods provides robust validation. In published studies, siRNA knockdown followed by immunoblotting has effectively demonstrated specificity of HAS3 antibodies, showing 70% decreased band intensity in silenced samples compared to controls .

What are the optimal sample preparation methods for detecting HAS3 in different applications?

Sample preparation varies by application and tissue type:

For Western Blotting:

• Prepare whole cell lysates using buffer containing: 50mM Tris-HCl (pH 7.5), 150mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol with protease inhibitors (10µg/ml leupeptin, 10µg/ml PMSF, 1µg/ml aprotinin, 1mM sodium vanadate, 5mM sodium fluoride)

• Load 50µg protein samples for electrophoresis

• Use 3.7% formaldehyde fixation for immunocytochemistry

For Immunohistochemistry:

• Perform antigen retrieval with TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Block with 25% normal goat serum to reduce background

For Flow Cytometry:

• Single-cell suspensions should be fixed appropriately based on target localization (membrane-associated versus intracellular HAS3)

How should I design experiments to study HAS3 function through antibody-based methods?

When designing HAS3 functional studies:

• Expression Analysis:

  • Compare HAS3 levels across normal versus pathological tissues using validated antibodies

  • Quantify via Western blot densitometry, normalized to housekeeping proteins

• Localization Studies:

  • Use immunocytochemistry with fluorescent secondary antibodies (e.g., Alexa Fluor 594) and counterstain with phalloidin to visualize actin

  • Examine subcellular localization as HAS3 is a multi-pass transmembrane protein

• Functional Assessment:

  • Combine antibody detection with particle exclusion assays (PEA) to measure pericellular HA matrix formation

  • Use Streptomyces hyaluronidase as an enzyme control to degrade HA

• Knockdown Validation:

  • Verify HAS3 silencing at both mRNA level (RT-PCR) and protein level (Western blot) using specific antibodies

  • Assess functional consequences through tumor growth assays

What controls should be included when working with HAS3 antibodies?

A comprehensive control strategy includes:

Positive Controls:

• Human fetal lung, kidney, and uterus lysates, which express detectable HAS3 levels

• Cell lines with confirmed HAS3 expression: PC-3, L02, HCT116

Negative Controls:

• Primary antibody omission to assess secondary antibody specificity

• Isotype-matched irrelevant antibody controls

• HAS3-knockdown samples (siRNA-treated)

Enzymatic Controls:

• Hyaluronidase treatment to confirm hyaluronan-dependent effects

Loading/Staining Controls:

• β-actin for Western blot normalization

• GAPDH for RT-PCR when validating knockdown

How can HAS3 antibodies be used to study cancer progression mechanisms?

HAS3 antibodies enable multiple approaches to study cancer progression:

• Tumor Growth and Metastasis Analysis:

  • Immunohistochemical assessment of HAS3 expression in primary versus metastatic tumors

  • Correlation of HAS3 levels with patient outcomes and clinicopathological features

• Mechanistic Studies:

  • Investigation of apoptotic pathways affected by HAS3 inhibition using caspase-3 staining in conjunction with HAS3 antibodies

  • Evaluation of proliferation markers alongside HAS3 expression

• Therapeutic Target Validation:

  • Assessment of HAS3 inhibition via small molecules like sorafenib (SF) using antibodies to quantify expression changes

  • Monitoring of HAS3 downregulation in dose-response studies, showing >80% decrease at 20 µM SF

Research has demonstrated that HAS3 inhibition decreases subcutaneous tumor growth by nearly 50% in colon cancer models, primarily by increasing apoptosis, as confirmed using HAS3 antibodies for expression verification .

How do I interpret multiple HAS3 protein bands in Western blot analyses?

Multiple HAS3 bands (single, doublet, or triplet) in immunoblots represent a complex but interpretable pattern:

• Structural Considerations:

  • HAS3 is a multi-pass transmembrane protein existing within lipid complexes

  • Multiple bands likely represent different protein states rather than non-specific binding

• Potential Interpretations:

  • Higher molecular weight bands: Protein complexes or post-translationally modified forms

  • Main band: Mature 63 kDa protein

  • Lower bands: Proteolytic fragments or biosynthetic precursors

• Validation Approaches:

  • Perform deglycosylation experiments to assess contribution of glycosylation

  • Use multiple antibodies targeting different epitopes to confirm band identity

  • Compare patterns across different cell types and treatments

Research indicates these multiple bands are closely related to HAS3 protein based on their response to HAS3-specific siRNA treatment and correlation with functional outcomes .

What methodologies combine HAS3 antibody detection with functional hyaluronan analysis?

Integration of antibody-based and functional approaches provides comprehensive insight:

• Particle Exclusion Assay (PEA):

  • Culture cells in 48-well plates

  • Treat with sodium acetate in phenol red-free MEM+0.1% BSA or Streptomyces hyaluronidase (control)

  • Incubate with 2mg/ml aggrecan in MEM/0.1% BSA

  • Add fixed erythrocytes to visualize exclusion zone

  • Correlate with HAS3 antibody staining in parallel samples

• Semi-quantitative RT-PCR with Protein Validation:

  • Isolate mRNA using Oligotex mRNA isolation kit

  • Perform RT-PCR with HAS3-specific primers

  • Normalize to GAPDH expression

  • Correlate with protein levels via Western blot using HAS3 antibodies

• In vivo Tumor Growth Combined with Molecular Analysis:

  • Generate stable HAS3-knockdown cancer cells

  • Implant subcutaneously in mice

  • Monitor tumor growth

  • Analyze harvested tumors for HAS3 expression, apoptosis (caspase-3), and proliferation markers

  • Correlate molecular changes with growth phenotypes

What are common challenges when using HAS3 antibodies and how can they be addressed?

How can I optimize HAS3 antibody detection in tissues with low expression levels?

For detecting low HAS3 expression:

• Signal Amplification Methods:

  • Employ tyramide signal amplification systems

  • Use biotin-streptavidin amplification for IHC

  • Consider more sensitive detection reagents (e.g., SuperSignal West Femto)

• Sample Enrichment:

  • Perform subcellular fractionation to concentrate membrane fractions where HAS3 localizes

  • Use immunoprecipitation to concentrate HAS3 before Western blotting

• Protocol Optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Adjust detergent concentration in lysis buffers to improve extraction

  • Optimize antigen retrieval conditions for IHC (test both pH 6.0 and pH 9.0 buffers)

• Antibody Selection:

  • Choose antibodies with demonstrated sensitivity in similar applications

  • Consider recombinant monoclonal antibodies for consistent performance

How should results from different HAS3 antibodies be compared and integrated?

When working with multiple HAS3 antibodies:

• Epitope Mapping:

  • Determine targeted regions (e.g., C-terminal region vs. full-length protein)

  • Antibodies targeting different epitopes may yield complementary information

• Systematic Validation:

  • Test each antibody under identical conditions with the same controls

  • Document differences in sensitivity, specificity, and binding patterns

• Cross-validation Strategy:

  • Use at least two antibodies from different sources or targeting different epitopes

  • Compare results with functional assays (e.g., hyaluronan production)

  • Correlate with mRNA expression data

• Standardization for Quantitative Comparisons:

  • Establish standard curves using recombinant HAS3

  • Use digital image analysis with consistent acquisition parameters

  • Apply the same normalization methods across experiments

How do HAS3 expression patterns correlate with biological significance in different tissues?

HAS3 expression varies across tissues with distinct biological implications:

Research indicates that HAS3 upregulation in metastatic SW620 colon cancer cells compared to primary tumor-derived SW480 cells suggests its role in cancer progression . Inhibition of HAS3 in these systems leads to decreased tumor growth, primarily through increased apoptosis rather than reduced proliferation .

How should researchers interpret contradictory results regarding HAS3 detection in the literature?

When encountering contradictory HAS3 research findings:

What current controversies exist in HAS3 research that antibody-based studies might help resolve?

Key controversies where HAS3 antibodies contribute to resolution:

• Functional Differences Between HAS Isoforms:

  • HAS3 produces lower molecular weight HA than HAS1/HAS2

  • Antibody-based studies can help delineate isoform-specific roles by enabling precise localization and quantification

• Mechanism of HAS3-Mediated Cancer Promotion:

  • Some studies suggest anti-apoptotic effects without impacting proliferation

  • Others indicate cell-type specific effects on both processes

  • Antibody-based detection of pathway components helps resolve these differences

• Regulatory Mechanisms of HAS3 Expression:

  • IFN-γ markedly upregulates while TGF-β downregulates HAS3 mRNA

  • Sorafenib downregulates HAS3 at concentrations ≥10 μM

  • Antibody detection enables precise quantification of these regulatory effects

• Therapeutic Targeting Approaches:

  • Direct HAS3 inhibition versus targeting upstream regulators

  • Antibody-based methods help validate mechanism of action for potential therapeutics

  • Correlation of expression levels with clinical outcomes guides therapeutic strategies

Antibody-based research has revealed that HAS3 inhibition increases apoptosis in colon cancer models, suggesting this as a primary mechanism for tumor growth reduction, rather than effects on proliferation .