st6gal2 Antibody

What is ST6GAL2 and what is its function in cellular processes?

ST6GAL2 (ST6 beta-galactosamide alpha-2,6-sialyltranferase 2) is a glycosyltransferase that transfers sialic acid from the donor substrate CMP-sialic acid to galactose-containing acceptor substrates. It specifically has alpha-2,6-sialyltransferase activity toward oligosaccharides with the Gal-beta-1,4-GlcNAc sequence at the non-reducing end of their carbohydrate groups. Unlike its more ubiquitous counterpart ST6GAL1, ST6GAL2 has restricted expression and demonstrates weak or no activity toward glycoproteins and glycolipids . The enzyme is primarily localized to the Golgi apparatus, consistent with other glycosyltransferases .

What are the structural characteristics of ST6GAL2 protein?

ST6GAL2 is a 529 amino acid (aa) type II transmembrane Golgi protein with a molecular weight of approximately 60-62 kDa. The protein contains an 11 aa N-terminal cytoplasmic region and a 497 aa luminal domain (aa 33-529) . When detecting ST6GAL2 via Western blot, researchers typically observe a band at approximately 60 kDa, which corresponds to the predicted molecular weight of the full-length protein .

How does ST6GAL2 differ from ST6GAL1 in terms of expression and function?

While both enzymes are sialyltransferases that add α2,6-linked sialic acids to terminal galactose residues, they differ significantly in their expression patterns and substrate preferences:

ST6GAL2 likely has additional biological functions beyond sialic acid transfer, as suggested by its enzymatic kinetics .

How can I validate the specificity of an ST6GAL2 antibody?

Validating ST6GAL2 antibody specificity requires a multi-technique approach:

• Genetic approaches: Use cell lines with genetic deletion of ST6GAL2 (e.g., ΔST6 cells) and compare with wild-type cells. Additionally, create rescue models by reexpressing ST6GAL2 in knockout lines to confirm restoration of antibody binding .

• Multiple detection methods: Employ various techniques including Western blot, immunocytochemistry/immunohistochemistry, and flow cytometry to ensure consistent results across different platforms .

• Size verification: For Western blots, confirm that the detected band appears at the expected molecular weight (~60 kDa) .

• Cellular localization pattern: ST6GAL2 should show a punctate, perinuclear Golgi-localization pattern in immunofluorescence studies, consistent with its known subcellular localization .

• Positive and negative tissue controls: Use tissues known to express or lack ST6GAL2 (e.g., brain tissue is positive, while certain other tissues may be negative) .

Why might antibodies advertised as ST6GAL2 antibodies show different staining patterns?

Differences in staining patterns between various ST6GAL2 antibodies may occur due to:

• Epitope differences: Antibodies raised against different regions of ST6GAL2 may have varying accessibility to the target depending on protein conformation or post-translational modifications.

• Antibody format: Polyclonal antibodies detect multiple epitopes and may show different patterns than monoclonal antibodies that target a single epitope.

• Cross-reactivity: Some antibodies may cross-react with related proteins like ST6GAL1 or other glycosyltransferases, especially if they share conserved domains.

• Detection of the product rather than the enzyme: Similar to confusion with ST6GAL1/CD75, some antibodies might recognize the sialic acid product rather than the ST6GAL2 enzyme itself .

Researchers should carefully examine the immunogen sequence and validation data provided by manufacturers and conduct their own validation experiments when possible .

What are the optimal conditions for detecting ST6GAL2 by Western blot?

For optimal Western blot detection of ST6GAL2:

• Sample preparation: Use fresh cell lysates or tissue homogenates, preferably with protease inhibitors to prevent degradation.

• Protein loading: Load 25-50 μg of total protein per lane for adequate detection .

• Recommended dilutions: Use antibody dilutions between 1:300-1:800, depending on the specific antibody and sample. The Proteintech antibody (28367-1-AP) recommends 1:300-1:800 for Western blot applications .

• Running conditions: Use reducing conditions and standard SDS-PAGE protocols. For example, R&D Systems detected ST6GAL2 using Immunoblot Buffer Group 1 .

• Detection system: HRP-conjugated secondary antibodies with ECL detection systems work well for ST6GAL2 detection .

• Expected band size: Look for a specific band at approximately 60 kDa, which corresponds to the full-length ST6GAL2 protein .

What is the recommended protocol for immunohistochemical detection of ST6GAL2?

For immunohistochemical detection of ST6GAL2:

• Sample preparation: Use fixed, paraffin-embedded tissue sections. R&D Systems recommends immersion-fixed paraffin-embedded sections .

• Antigen retrieval: Perform heat-induced epitope retrieval using basic antigen retrieval reagents (e.g., R&D Systems Antigen Retrieval Reagent-Basic, CTS013) .

• Antibody concentration: For R&D Systems antibody, use 15 μg/mL incubated overnight at 4°C . For HPA014459, use dilutions between 1:50-1:200 .

• Detection system: HRP-DAB systems work well, such as Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS019) .

• Counterstaining: Hematoxylin counterstaining helps visualize tissue architecture .

• Expected pattern: Look for specific staining in the cytoplasm of target cells, particularly in neurons when examining brain tissue .

How do I quantify ST6GAL2 expression in experimental samples?

Quantification of ST6GAL2 expression can be performed through several methods:

• Western blot densitometry: Normalize ST6GAL2 band intensity to loading controls (e.g., GAPDH, β-actin) using image analysis software. In knockdown experiments, expression was quantified as a percentage reduction compared to control groups (e.g., 36.7% ± 0.028% in MCF-7 cells) .

• Immunohistochemistry scoring: For tissue samples, categorize expression levels based on percentage of positive cells:

  • High expression: ≥25% of tumor cells with positive staining

  • Low expression: <25% of tumor cells with positive staining

• Real-time PCR: For mRNA quantification, use established primers and normalize to housekeeping genes. This approach confirmed ST6GAL2 overexpression in breast cancer tissues compared to adjacent normal tissues .

• Flow cytometry: For cell surface sialylation products, mean fluorescence intensity can be quantified, though this detects the product rather than the enzyme itself .

How does ST6GAL2 expression relate to cancer progression?

ST6GAL2 has been implicated in cancer biology with variable expression patterns across different cancer types:

• Breast cancer: ST6GAL2 is overexpressed in breast cancer tissues compared to adjacent normal tissues. Higher expression correlates with advanced tumor stage and poorer survival outcomes .

• Survival impact: Patients with lower ST6GAL2 expression in breast cancer lived notably longer than those with higher expression .

• Cellular mechanisms: Silencing ST6GAL2 inhibits cancer progression by:

  • Arresting cell cycle progression at the G0/G1 phase

  • Inhibiting cell adhesion and invasion

  • Reducing expression of adhesion molecules (ICAM-1, VCAM-1, CD24) and metastasis-related proteins (MMP2, MMP9, CXCR4)

• Pathway associations: ST6GAL2 positively correlates with focal adhesion and metastasis pathways in cancer cells .

• In vivo effects: ST6GAL2 downregulation reduces tumor growth in mouse xenograft models, with significantly smaller tumor volumes compared to control tumors .

What methodological approaches can be used to study ST6GAL2 function in disease models?

To investigate ST6GAL2 function in disease models, researchers have employed several methodological approaches:

• Gene silencing: Lentiviral transduction to knockdown ST6GAL2 expression, followed by functional assays to assess effects on cellular behaviors .

• Cell viability assays: CCK-8 assays to measure proliferation at different time points (0, 12, 24, 48, and 72 hours) after ST6GAL2 modulation .

• Cell cycle analysis: Flow cytometry to analyze cell cycle distribution (G0/G1, S, G2/M phases) following ST6GAL2 silencing .

• Xenograft models: Subcutaneous injection of ST6GAL2-modulated cells into nude mice with subsequent tumor volume measurements over time .

• Gene expression correlation: Bioinformatic analyses to identify pathways and genes correlated with ST6GAL2 expression .

• Immunoblotting for downstream targets: Western blot analysis of adhesion molecules and metastasis-related proteins to identify mechanisms of ST6GAL2 action .

How does ST6GAL2 expression correlate with clinical parameters in cancer patients?

Studies examining ST6GAL2 expression in relation to clinical parameters have revealed:

• Tumor stage correlation: ST6GAL2 expression is significantly associated with tumor stage in breast cancer patients .

• Receptor status: ST6GAL2 expression correlates with ER/PR/HER2 status in breast cancer patients, suggesting potential hormone-dependent regulation .

• Prognostic value: Lower ST6GAL2 expression is associated with longer survival time in breast cancer patients .

• Correlation with clinical parameters: The table below summarizes clinical correlations found in breast cancer studies:

These findings suggest ST6GAL2 could serve as a potential prognostic marker and therapeutic target in cancer treatment .

How can I distinguish between antibodies that recognize ST6GAL2 versus its sialylation products?

Distinguishing between antibodies that detect the ST6GAL2 enzyme versus its sialylation products requires careful experimental design:

• Subcellular localization: ST6GAL2 enzyme is primarily localized in the Golgi apparatus, while its sialylation products are found on cell surfaces and secreted proteins. Use cell fractionation and immunofluorescence microscopy to distinguish these locations .

• Neuraminidase treatment: Treat samples with neuraminidase enzymes that cleave sialic acids. If antibody binding is reduced after treatment, it likely recognizes sialylated products rather than the enzyme itself .

• Flow cytometry of non-permeabilized cells: ST6GAL2 enzyme should not be detectable on the surface of non-permeabilized cells, while sialylated products would be accessible. This approach revealed that some purported "ST6GAL1" antibodies were actually detecting surface sialylation (CD75 epitope) .

• Knockout model comparison: Use genetic models with ST6GAL2 deletion and observe whether antibody reactivity is lost in immunoblotting, immunohistochemistry, and flow cytometry. True ST6GAL2 antibodies should show no reactivity in knockout models .

• Size verification: In Western blots, antibodies detecting the enzyme should recognize a band at ~60 kDa, while those detecting sialylated products may show multiple bands of various sizes .

What are the potential pitfalls when interpreting ST6GAL2 antibody results across different experimental systems?

Researchers should be aware of several potential pitfalls when working with ST6GAL2 antibodies:

• Antibody cross-reactivity: Some antibodies may cross-react with the related enzyme ST6GAL1 due to sequence similarities. This issue has created significant confusion in the field, particularly with antibodies originally developed against CD75 epitopes .

• Variable glycosylation states: ST6GAL2 itself is glycosylated, and its glycosylation pattern may vary between cell types and disease states, potentially affecting antibody recognition.

• Tissue-specific expression levels: ST6GAL2 has restricted expression, primarily in neurons and bronchial epithelium, so sensitivity may be an issue in tissues with low expression levels .

• Post-translational modifications: Different fixation or sample preparation methods may affect post-translational modifications and consequently epitope accessibility.

• Non-specific bands in Western blots: Several ST6GAL2 antibodies produce multiple bands in Western blots. Careful validation with positive and negative controls is essential to identify the specific band .

• Database inconsistencies: The Human Protein Atlas and other databases may contain data generated using insufficiently validated antibodies, leading to confusion about actual expression patterns .

How should I design experiments to study the causal relationship between ST6GAL2 expression and disease progression?

To establish causal relationships between ST6GAL2 and disease progression, consider these experimental approaches:

• Gene modulation strategies:

  • Knockdown: Use shRNA or siRNA to reduce ST6GAL2 expression and assess functional consequences

  • Overexpression: Generate stable cell lines overexpressing ST6GAL2 to examine gain-of-function effects

  • CRISPR/Cas9: Create complete knockout cell lines for more definitive functional studies

• Rescue experiments: After knocking down ST6GAL2, reintroduce either wild-type or mutant forms (e.g., catalytically inactive) to determine which protein domains are essential for the observed phenotypes .

• Animal models:

  • Xenograft models: Inject cells with modified ST6GAL2 expression into immunocompromised mice

  • Transgenic models: Generate tissue-specific ST6GAL2 knockout or overexpression mouse models

• Clinical correlations:

  • Analyze patient tissues for ST6GAL2 expression in relation to:

    • Disease stage and progression

    • Treatment response

    • Survival outcomes

  • Use multivariate analysis to control for confounding factors

• Mechanistic studies:

  • Identify downstream targets and pathways using techniques like RNA-Seq

  • Examine changes in global sialylation patterns using lectin arrays or mass spectrometry

  • Investigate protein-protein interactions using co-immunoprecipitation

These comprehensive approaches can help establish whether ST6GAL2 is merely a biomarker or a driver of disease processes, potentially identifying new therapeutic targets .