ST6GALNAC5 Antibody

What is ST6GALNAC5 and what is its biological significance?

ST6GALNAC5 (ST6 GalNAc alpha-2,6-sialyltransferase V) is a sialyltransferase enzyme involved in the biosynthesis of gangliosides, particularly GD1a from GM1b. Gangliosides are acidic glycosphingolipids containing sialic acid residues that play crucial modulatory roles in cellular recognition, interaction, adhesion, and signal transduction . ST6GALNAC5 expression is predominantly restricted to brain tissue, where it contributes to the developmental regulation of gangliosides in the nervous system . The enzyme has also been identified as a key mediator in breast cancer metastasis to the brain, potentially facilitating cancer cell crossing of the blood-brain barrier . Understanding ST6GALNAC5 function is critical for both neurodevelopmental research and cancer metastasis investigations, as it represents a potential therapeutic target for preventing brain metastasis.

What are the key characteristics of commercially available ST6GALNAC5 antibodies?

Current ST6GALNAC5 antibodies are predominantly mouse monoclonal antibodies developed against specific regions of the human ST6GALNAC5 protein. The two main clones available are #719508 and #719526, both recognizing the region spanning Gly30-Phe336 of the human protein (Accession # Q9BVH7) . These antibodies demonstrate high specificity, with no cross-reactivity observed with related sialyltransferases such as recombinant human ST6GALNAC4 or ST6GALNAC6 in direct ELISA applications . They are typically supplied as lyophilized preparations from filtered PBS solutions with trehalose, requiring reconstitution before use. Small pack sizes (SP) may be supplied as filtered solutions in PBS without preservatives . These antibodies have been validated for different applications, with clone #719508 optimized for immunocytochemistry and clone #719526 validated for Western blot applications where it detects a specific band at approximately 36 kDa under reducing conditions .

How does ST6GALNAC5 function differ from other sialyltransferases?

ST6GALNAC5 belongs to the ST6GalNAc subfamily of sialyltransferases but demonstrates distinct substrate specificity and tissue distribution compared to related enzymes. While many sialyltransferases have broad expression patterns, ST6GALNAC5 expression is largely restricted to brain tissue, indicating specialized neurological functions . Unlike ST6GALNAC4 and ST6GALNAC6, which may act on different glycolipid and glycoprotein substrates, ST6GALNAC5 specifically catalyzes the transfer of sialic acid to the GalNAc residue of GM1b ganglioside to form GD1a . This substrate specificity makes ST6GALNAC5 crucial for the proper composition of brain gangliosides during development. The distinct epitope recognition profile of ST6GALNAC5 antibodies, which show no cross-reactivity with ST6GALNAC4 or ST6GALNAC6, further confirms these structural and functional differences . Understanding these distinctions is essential for researchers designing targeted experiments to investigate ganglioside metabolism in normal brain function and pathological conditions.

What are the optimal conditions for using ST6GALNAC5 antibodies in immunocytochemistry?

For optimal immunocytochemistry results with ST6GALNAC5 antibodies, researchers should employ the following protocol: Use the mouse monoclonal anti-human ST6GALNAC5 antibody (clone #719508) at a concentration range of 8-25 μg/mL, with 10 μg/mL being effective for many applications . Incubate with cells for 3 hours at room temperature after appropriate fixation (immersion fixation protocols work well with MDA-MB-231 cells) . For detection, employ fluorescently-labeled secondary antibodies such as NorthernLights 557-conjugated Anti-Mouse IgG at manufacturer-recommended dilutions . Include DAPI counterstaining to visualize nuclei and provide cellular context. Based on published results, expect to observe specific staining localized to cell membranes and cytoplasm in positive samples . To ensure specificity, include appropriate negative controls (primary antibody omission) and positive controls (MDA-MB-231 cells are recommended as they express detectable levels of ST6GALNAC5) . For multi-labeling experiments, carefully select complementary fluorophores to avoid spectral overlap, and perform sequential incubations if cross-reactivity between antibodies is a concern.

How should researchers optimize Western blot protocols for ST6GALNAC5 detection?

For effective Western blot detection of ST6GALNAC5, researchers should follow this optimized protocol: Use clone #719526 antibody at a concentration of 2 μg/mL for probing PVDF membranes . Prepare cell/tissue lysates under reducing conditions using appropriate buffer systems (Immunoblot Buffer Group 1 has been validated) . Resolve proteins using standard SDS-PAGE (10-12% gels are recommended for detecting the approximately 36 kDa ST6GALNAC5 protein) . For detection, use HRP-conjugated anti-mouse IgG secondary antibodies followed by enhanced chemiluminescence. When interpreting results, look for a specific band at approximately 36 kDa, which represents the ST6GALNAC5 protein . MDA-MB-231 breast cancer cell lysates serve as excellent positive controls . For challenging samples with low expression, consider enrichment steps such as immunoprecipitation before Western blotting. If multiple bands appear, validate specificity through knockout/knockdown controls or peptide competition assays. Researchers should also be aware that post-translational modifications like glycosylation might affect the apparent molecular weight of ST6GALNAC5 in some tissue samples.

What considerations are important when designing qPCR experiments to correlate with ST6GALNAC5 protein expression?

When designing qPCR experiments to complement ST6GALNAC5 protein analysis, researchers should implement the following methodological approach: First, design primers spanning exon-exon junctions of the ST6GALNAC5 gene to avoid genomic DNA amplification. Reference the human ST6GALNAC5 accession information (such as Q9BVH7) to identify appropriate target regions . Validate primers using standard curves with efficiency between 90-110% and single-peak melt curves. Select appropriate reference genes based on the experimental context—for brain tissue research, genes like GAPDH, ACTB, and tissue-specific stable references should be evaluated. For breast cancer metastasis studies, include multiple reference genes validated in breast cancer models. Extract RNA using methods that preserve integrity, confirmed by RIN values >7. Perform reverse transcription with consistent input RNA amounts across all samples. Run qPCR reactions in triplicate with no-template and no-RT controls. When analyzing data, use the comparative Ct method (2^-ΔΔCt) with appropriate normalization to reference genes. Crucially, correlate mRNA and protein expression through parallel samples, recognizing that post-transcriptional regulation may lead to discrepancies between mRNA and protein levels of ST6GALNAC5.

How can ST6GALNAC5 antibodies be utilized to study breast cancer brain metastasis mechanisms?

To investigate breast cancer brain metastasis mechanisms using ST6GALNAC5 antibodies, researchers should implement a multi-faceted experimental approach: Begin with comparative immunohistochemistry analysis of matched primary breast tumors and brain metastases using validated ST6GALNAC5 antibodies (clone #719508 at 10 μg/mL) . Quantify expression differences using digital pathology and correlate with clinical outcomes. Establish in vitro blood-brain barrier (BBB) models using brain microvascular endothelial cells and astrocytes to test how ST6GALNAC5 expression influences cancer cell transmigration . Use MDA-MB-231 cells as a model system, as these have demonstrated ST6GALNAC5 expression and brain metastatic potential . Employ both gain-of-function (overexpression) and loss-of-function (shRNA/CRISPR) approaches to modify ST6GALNAC5 levels in breast cancer cell lines, then assess changes in invasive behavior and BBB penetration. Analyze ST6GALNAC5-mediated changes in cell surface ganglioside composition using specific ganglioside antibodies alongside ST6GALNAC5 detection. For in vivo studies, use orthotopic xenograft models with modified ST6GALNAC5 expression and monitor brain metastasis development using bioluminescence imaging and histological analysis. This comprehensive approach will help delineate the specific mechanisms by which ST6GALNAC5 facilitates breast cancer brain metastasis, potentially identifying intervention points.

What approaches can be used to study the role of ST6GALNAC5 in neurodevelopment and neurological disorders?

To investigate ST6GALNAC5's role in neurodevelopment and neurological disorders, researchers should implement this methodological framework: Begin with developmental expression profiling of ST6GALNAC5 in brain tissue across different developmental stages using Western blotting (clone #719526) and immunohistochemistry (clone #719508) . Perform co-localization studies with neuronal, glial, and progenitor cell markers to determine cell type-specific expression patterns. Utilize primary neural cultures or brain organoids to study the temporal regulation of ST6GALNAC5 during neural differentiation. Apply CRISPR/Cas9 gene editing or RNAi techniques to modulate ST6GALNAC5 expression, then assess effects on neurite outgrowth, synaptogenesis, and neuronal migration. Employ lipidomic approaches to correlate ST6GALNAC5 expression levels with specific changes in ganglioside profiles, particularly GD1a synthesis from GM1b . For neurological disorder studies, analyze ST6GALNAC5 expression in patient-derived samples (when available) or relevant animal models of conditions where ganglioside metabolism is implicated, such as neurodegenerative diseases or developmental disorders. Functional assays should include electrophysiological measurements to determine if ST6GALNAC5-mediated ganglioside synthesis affects neuronal excitability or synaptic transmission. This integrated approach will illuminate ST6GALNAC5's contributions to normal brain development and potential roles in neurological pathologies.

How can multiplexed imaging techniques be optimized for studying ST6GALNAC5 in heterogeneous tissue samples?

For optimizing multiplexed imaging of ST6GALNAC5 in heterogeneous tissues, researchers should implement this technical strategy: Begin with tissue preparation optimization—for brain or tumor samples, use neutral-buffered formalin fixation with controlled fixation times (12-24 hours) followed by careful antigen retrieval protocols to preserve both ST6GALNAC5 epitopes and other target antigens. For multiplex immunofluorescence, use clone #719508 antibody at 10 μg/mL alongside antibodies against relevant cell type markers or signaling pathway components . To avoid cross-reactivity, employ antibodies from different host species or use direct conjugates. When species conflicts are unavoidable, implement sequential staining with complete stripping or blocking between rounds. For sequential multiplexing approaches like Cyclic Immunofluorescence (CycIF) or CODEX, validate that the ST6GALNAC5 epitope remains stable through multiple stripping cycles. For highly complex panels, consider tyramide signal amplification to detect low-abundance ST6GALNAC5 expression while minimizing antibody cross-talk. Incorporate spectral unmixing algorithms during image acquisition and analysis to resolve overlapping fluorophores. For spatial analysis, implement digital pathology tools to quantify ST6GALNAC5 expression patterns relative to anatomical features and other markers. Validate findings with orthogonal methods such as spatial transcriptomics or laser capture microdissection followed by molecular analysis. This comprehensive approach enables detailed characterization of ST6GALNAC5 expression in complex tissue environments like brain metastases or developing neural tissues.

How should researchers address inconsistent ST6GALNAC5 antibody staining patterns?

When encountering inconsistent ST6GALNAC5 antibody staining, researchers should systematically troubleshoot using this approach: First, verify antibody quality by testing new aliquots and confirming proper storage (avoid repeated freeze-thaw cycles) . Examine fixation conditions, as overfixation can mask epitopes—test different fixation durations (10-30 minutes with 4% paraformaldehyde) and antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0 at 95-100°C for 10-20 minutes). Optimize antibody concentration by performing titration experiments across a range (5-25 μg/mL) with positive control MDA-MB-231 cells . Extend primary antibody incubation time (overnight at 4°C may improve signal-to-noise ratio compared to 3 hours at room temperature). Evaluate permeabilization protocols—ST6GALNAC5's localization to both membrane and cytoplasmic compartments requires balanced permeabilization (0.1-0.3% Triton X-100 or 0.1% saponin) . For weak signals, implement amplification systems like tyramide signal amplification or high-sensitivity detection reagents. Address background issues by increasing blocking stringency (5% BSA or 10% serum from the same species as the secondary antibody) and extending blocking times (1-2 hours). Include validated positive controls (MDA-MB-231 cells) and negative controls (primary antibody omission or isotype controls) in each experiment . Finally, confirm specificity through siRNA knockdown validation in your specific cell system.

What factors might contribute to discrepancies between protein and mRNA expression levels of ST6GALNAC5?

Discrepancies between ST6GALNAC5 protein and mRNA levels can arise from multiple factors that researchers should consider during data interpretation: Post-transcriptional regulation through microRNAs may target ST6GALNAC5 mRNA—analyze miRNA databases for predicted miRNA binding sites on ST6GALNAC5 transcripts. Examine mRNA stability, as the half-life of ST6GALNAC5 mRNA may vary across cell types or under different conditions; perform actinomycin D chase experiments to determine mRNA decay rates. Consider translational efficiency variations, which can be assessed using polysome profiling to determine if ST6GALNAC5 mRNA is efficiently loaded onto ribosomes. Post-translational modifications may affect protein stability—treat cells with proteasome inhibitors (MG132) or lysosomal inhibitors (chloroquine) to assess degradation pathways. Evaluate protein half-life through cycloheximide chase experiments. Technical factors may also contribute, including differences in detection sensitivity between antibody-based protein detection and PCR-based mRNA quantification. Detection of alternative splice variants may occur—design PCR primers and use antibodies that recognize common regions or specific isoforms. Subcellular localization or compartmentalization might impact protein extraction efficiency—compare different protein extraction methods (RIPA, NP-40, or Triton X-100 based buffers). To address these issues, researchers should perform time-course experiments examining both mRNA and protein levels simultaneously under various experimental conditions.

How can researchers validate the specificity of ST6GALNAC5 antibody staining in tissue samples?

To validate ST6GALNAC5 antibody specificity in tissue samples, researchers should implement this comprehensive validation strategy: Perform side-by-side comparisons of multiple antibody clones (both #719508 and #719526) targeting different epitopes of ST6GALNAC5 . Use peptide competition assays, where pre-incubating the antibody with excess immunizing peptide (Gly30-Phe336 region of ST6GALNAC5) should abolish specific staining . Implement gene silencing approaches in appropriate model systems—compare staining patterns in wild-type and CRISPR/siRNA ST6GALNAC5-knockdown samples. For tissue studies, include known positive controls (brain tissue, MDA-MB-231 xenografts) and negative controls (tissues with documented low expression) . Apply orthogonal detection methods—correlate immunohistochemistry results with in situ hybridization for ST6GALNAC5 mRNA in sequential tissue sections. For brain metastasis research, compare primary tumors with matched brain metastases, as upregulation in metastatic sites would align with the known biology . Assess staining patterns for expected subcellular localization (membrane and cytoplasmic distribution in the Golgi apparatus where sialyltransferases function) . When possible, correlate antibody staining with functional assays—such as sialyltransferase activity using specialized enzymatic assays. Document all validation steps methodically, including images of positive and negative controls alongside experimental samples for publication.

What are the emerging research areas linking ST6GALNAC5 to neurological disorders beyond cancer?

Emerging research connecting ST6GALNAC5 to neurological disorders extends beyond its established role in cancer metastasis. Developmental neurobiology investigations are exploring how ST6GALNAC5-mediated ganglioside synthesis influences critical neurodevelopmental processes such as neuronal migration, axon guidance, and synaptogenesis . Given ST6GALNAC5's brain-restricted expression pattern and role in ganglioside synthesis, researchers are investigating potential connections to neurodegenerative disorders where ganglioside dysregulation has been implicated, including Parkinson's and Alzheimer's diseases . The enzyme's role in producing complex gangliosides may influence amyloid-beta processing and tau phosphorylation pathways. Neuroinflammatory conditions represent another promising research direction, as gangliosides mediate interactions between neurons and immune cells. Methodologically, researchers are employing conditional knockout models with brain region-specific deletion of ST6GALNAC5 to assess behavioral and pathological consequences. Advanced glycolipidomic approaches are being used to create comprehensive profiles of ganglioside changes in neurological disease models. Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons provide platforms for studying ST6GALNAC5 function in human neural cells with disease-relevant genetic backgrounds. These emerging research directions may reveal new therapeutic targets for neurological conditions through modulation of ST6GALNAC5 activity.

How might single-cell analysis techniques advance understanding of ST6GALNAC5 function in heterogeneous tissues?

Single-cell analysis techniques offer powerful approaches to decipher ST6GALNAC5 function in complex tissues through these methodological applications: Single-cell RNA sequencing can reveal cell type-specific expression patterns of ST6GALNAC5 across brain regions or within heterogeneous tumor microenvironments, identifying previously unrecognized expression in rare cell populations. Emerging single-cell proteomics approaches, though still technically challenging, may eventually enable detection of ST6GALNAC5 protein at the single-cell level. Single-cell glycomics, an emerging field, could potentially measure cell-specific ganglioside profiles correlated with ST6GALNAC5 expression. Spatial transcriptomics technologies like Visium or MERFISH can map ST6GALNAC5 expression within the anatomical context of brain tissue or tumor microenvironments, revealing spatial relationships to specific anatomical features. Mass cytometry (CyTOF) with metal-conjugated ST6GALNAC5 antibodies allows multiplexed protein analysis across large cell populations, capturing expression heterogeneity. For functional studies, CRISPR screens with single-cell readouts can identify genes that interact with ST6GALNAC5 in specific cellular contexts. Methodologically, researchers should optimize antibody concentrations for single-cell techniques, develop appropriate computational pipelines for data analysis, and integrate multiple single-cell modalities for comprehensive understanding. These approaches will reveal how ST6GALNAC5 expression varies across cell types and states, potentially identifying specialized functions in particular neural subpopulations or tumor cell subsets.

What therapeutic strategies might target ST6GALNAC5 for preventing brain metastasis?

Developing therapeutic strategies targeting ST6GALNAC5 to prevent brain metastasis requires a multi-faceted translational research approach: Small molecule inhibitor development represents an immediate opportunity—researchers should screen compound libraries for molecules that selectively inhibit ST6GALNAC5 enzymatic activity using biochemical assays with purified enzyme and appropriate ganglioside substrates . Structure-based drug design based on the catalytic domain (Gly30-Phe336) could yield selective inhibitors . Antibody-drug conjugates targeting ST6GALNAC5-expressing cancer cells offer another approach, leveraging the validated antibodies (clones #719508 and #719526) as targeting moieties linked to cytotoxic payloads . RNA interference strategies delivered via nanoparticles could suppress ST6GALNAC5 expression in circulating tumor cells before they establish brain metastases. Gene editing approaches using CRISPR/Cas9 technology delivered by appropriate vectors might permanently disable ST6GALNAC5 in metastatic cells. For all these approaches, researchers must establish appropriate models for efficacy testing, including blood-brain barrier penetration assays, mouse models of brain metastasis, and patient-derived organoids. Importantly, any therapeutic strategy must address the challenge of brain-specific ST6GALNAC5 expression—therapeutic approaches should ideally target cancer-specific features of ST6GALNAC5 expression or function to minimize disruption of normal neurological roles . Combination therapies targeting ST6GALNAC5 along with other mediators of brain metastasis may provide the most effective approach to preventing this devastating complication of breast cancer.