B3GNT6 Antibody

What is B3GNT6 and what is its biological function?

B3GNT6, also known as core 3 synthase, is a member of the Glycosyltransferase 31 protein family with a canonical length of 384 amino acid residues and molecular weight of approximately 42.7 kDa in humans. It functions as a Beta-1,3-N-acetylglucosaminyltransferase that synthesizes the core 3 structure of O-glycan, which serves as an important precursor in the biosynthesis of mucin-type glycoproteins. The enzyme catalyzes the addition of N-acetylglucosamine to N-acetylgalactosamine-modified serine or threonine residues. B3GNT6 is localized in the Golgi apparatus, consistent with its role in post-translational glycosylation pathways .

Which tissues predominantly express B3GNT6?

B3GNT6 shows tissue-specific expression patterns that correlate with its functional significance in mucin biosynthesis:

Understanding these expression patterns is crucial when selecting appropriate positive control tissues for antibody validation. For instance, colon tissue is frequently used as a positive control in Western blot applications due to its consistently high B3GNT6 expression .

What are the common applications for B3GNT6 antibodies?

B3GNT6 antibodies are utilized across multiple experimental techniques in glycobiology research:

When performing immunohistochemistry, suggested antigen retrieval conditions include using TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative. It is recommended to titrate antibody concentrations for each specific experimental system to achieve optimal signal-to-noise ratios .

How should I validate the specificity of B3GNT6 antibodies in my experimental system?

Antibody validation is critical for ensuring experimental rigor. For B3GNT6 antibodies, a multi-modal validation approach is recommended:

• Positive tissue controls: Use tissues known to express B3GNT6 (colon, small intestine, testis) as positive controls.

• Knockdown/knockout verification: CRISPR/Cas9-based knockdown systems can confirm antibody specificity. Published studies have employed this approach for glycosyltransferases including B3GNT6.

• Expected molecular weight confirmation: Verify detection at the expected molecular weight (~43 kDa).

• Cross-reactivity assessment: Test the antibody against related glycosyltransferases to ensure specificity.

• Multiple antibody comparison: Use antibodies from different sources/clones to confirm consistent detection patterns .

What are the critical parameters for optimizing Western blot detection of B3GNT6?

Successful Western blot detection of B3GNT6 requires careful optimization:

When working with weakly expressing samples, consider enrichment techniques such as immunoprecipitation prior to Western blot analysis. Additionally, evaluate antibody specificity using relevant knockdown/knockout controls to confirm the identity of detected bands .

How can I optimize immunohistochemical detection of B3GNT6 in tissue sections?

Effective immunohistochemical detection of B3GNT6 requires careful consideration of tissue processing and staining protocols:

• Tissue fixation: Standard formalin fixation (10% neutral buffered formalin for 24-48 hours) is generally compatible with B3GNT6 detection.

• Sectioning: 4 μm thick sections mounted on positively charged glass slides are recommended.

• Antigen retrieval: Heat-induced epitope retrieval using TE buffer (pH 9.0) is preferred, though citrate buffer (pH 6.0) may be used as an alternative.

• Antibody dilution: Begin with a 1:100 dilution and optimize between 1:50-1:500 based on signal intensity and background.

• Detection system: For rabbit-generated B3GNT6 antibodies, universal secondary antibody systems like the Impress reagent kit are suitable, followed by development with peroxidase kits.

• Scoring method: Implement a composite scoring system based on staining intensity (-/+/++/+++) and extent (percent positive cells), with final scores ranging from 0-12 for semi-quantitative analysis .

How should I design experiments to investigate B3GNT6 function in glycosylation pathways?

Effective experimental approaches to study B3GNT6 function include:

• Gene modulation strategies:

  • CRISPR/Cas9-based knockdown/knockout systems for loss-of-function studies

  • Overexpression systems using expression vectors containing the B3GNT6 cDNA

  • Inducible expression systems for temporal control of B3GNT6 expression

• Functional assessment methods:

  • Glycan profiling using lectin blots to detect changes in core 3 O-glycan structures

  • Mass spectrometry analysis of glycan patterns

  • Mucin immunoblotting to assess changes in glycosylation of specific mucins (e.g., MUC1, MUC4, MUC5AC, MUC5B)

• Phenotypic assays:

  • Cell proliferation assays (e.g., MTT assay) to assess growth effects

  • Migration assays to evaluate motility changes

  • Analysis of epithelial-mesenchymal transition (EMT) marker expression

  • Assessment of stem cell marker expression in cancer models

What are common challenges when working with B3GNT6 antibodies and how can they be addressed?

When experiencing weak or inconsistent results, consider using tissues with known high B3GNT6 expression (e.g., colon tissue, testis) as positive controls to establish optimal detection conditions .

What are the recommended positive and negative controls for B3GNT6 antibody experiments?

The use of appropriate controls is essential for result interpretation. In particular, CRISPR/Cas9-based knockdown systems have been successfully employed to validate antibody specificity for glycosyltransferases, including B3GNT6 .

How is B3GNT6 expression and function altered in disease states?

B3GNT6 expression has been studied in various pathological conditions, particularly in cancer. While search result focused more on other glycosyltransferases like B3GNT3 in pancreatic cancer, related research has investigated B3GNT6's role in various cancers:

• Altered expression: Changes in B3GNT6 expression have been observed in several cancer types, particularly in gastrointestinal cancers where mucin glycosylation patterns are altered.

• Functional significance: As the enzyme responsible for core 3 O-glycan synthesis, alterations in B3GNT6 can significantly impact mucin glycosylation patterns, potentially affecting cell adhesion, immune recognition, and metastatic potential.

• Biomarker potential: Expression patterns of B3GNT6 and its glycan products are being investigated as potential biomarkers for certain cancer types and stages.

Research approaches to study these alterations include immunohistochemical analysis of human cancer tissues compared to normal tissues, correlation of expression with clinical parameters, and functional studies using cell line models .

What methodologies are used to study B3GNT6 in cancer research models?

Cancer researchers employ several approaches to investigate B3GNT6's role:

• Expression analysis:

  • RT-qPCR for mRNA quantification

  • Western blot for protein detection

  • Immunohistochemistry for tissue localization and semi-quantitative analysis

• Functional studies:

  • CRISPR/Cas9-based gene editing for knockdown/knockout

  • Proliferation assays (e.g., MTT assay conducted over 7 days)

  • Migration and invasion assays

  • Colony formation assays

  • Analysis of EMT markers (E-cadherin, ZO1, Zeb1, Snail)

  • Assessment of stem cell markers (OCT3/4, SOX2, SOX9, CD44)

• Glycan analysis:

  • Lectin blots to detect specific glycan structures

  • Mass spectrometry for comprehensive glycan profiling

  • Immunoblotting for mucins that are modified by B3GNT6 (e.g., MUC4)

• Pathway analysis:

  • Proteomics to identify altered protein expression patterns

  • Investigation of specific signaling pathways (e.g., β-catenin)

How can B3GNT6 antibodies be integrated with other techniques for comprehensive glycobiology studies?

Integrating B3GNT6 antibody-based detection with complementary techniques provides a more comprehensive understanding of glycosylation biology:

• Multi-omics approaches:

  • Combine B3GNT6 expression analysis (using antibodies) with transcriptomics data (RNA-Seq)

  • Correlate with glycomics data (mass spectrometry-based glycan profiling)

  • Integrate with proteomics data to identify glycoprotein substrates

• Functional glycomics:

  • Use B3GNT6 antibodies in conjunction with lectin arrays to correlate enzyme expression with glycan profiles

  • Combine with CRISPR/Cas9 editing to establish cause-effect relationships

• Structural biology integration:

  • Correlate B3GNT6 expression/localization with structural characterization of glycans

  • Use with glycan-specific antibodies to detect specific O-glycan structures

• Translational approaches:

  • Combine IHC detection of B3GNT6 with patient outcome data for biomarker studies

  • Correlate with functional assays in patient-derived models

These integrated approaches provide more robust and comprehensive insights into the role of B3GNT6 in normal physiology and disease states .