B3GNT6 antibodies are immunodetection reagents designed to bind specifically to the B3GNT6 enzyme. These antibodies are used to:
Quantify protein expression in tissues (e.g., colon, stomach) via Western blot (WB) or immunohistochemistry (IHC) .
Investigate post-translational modifications and glycosylation pathways .
Study disease mechanisms, including cancer progression and immune disorders .
Colorectal and Gastric Cancers: B3GNT6 is downregulated in tumor tissues compared to normal tissues. Low expression correlates with poor survival, KRAS mutations, and chromosomal instability .
Pancreatic Cancer: B3GNT6 mRNA stability is enhanced by IGF2BP2 via m6A methylation, promoting tumor progression .
Prostate Cancer: Overexpression reduces tumor metastasis in murine models .
Selective IgA Deficiency (IgAD): A genome-wide association study identified B3GNT6 as a susceptibility gene in patients lacking HLA risk alleles .
Glycosylation Role: B3GNT6 synthesizes the core 3 O-glycan structure (GlcNAc-β1,3-GalNAc-α1-Ser/Thr), critical for mucin biosynthesis .
Pathway Enrichment: Low B3GNT6 levels are linked to upregulated proteasome activity and KRAS/ERK signaling in colorectal cancer .
Application | Dilution Range |
---|---|
Western Blot (WB) | 1:500–1:2000 |
IHC | 1:50–1:500 |
WB: Use RIPA buffer for extraction; detect with chemiluminescence .
IHC: Antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
Biomarker Potential: B3GNT6 expression serves as a diagnostic marker for colorectal cancer (AUC = 0.95 in GSE39582 dataset) .
Therapeutic Target: Inhibition of B3GNT6-regulating pathways (e.g., IGF2BP2/m6A axis) may suppress pancreatic cancer progression .
Applications : Co-immunoprecipitation (Co-IP)
Sample type: cells
Review: Furthermore, immunoprecipitation experiments on LOVO cells transfected with the pcDNA3.1-B3GNT6 vector showed that B3GNT6 can directly interact with MUC2 (Figure 2.G). The BSL-II lectin staining demonstrated that overexpression of B3GNT6 caused a significant increase in the level of Core3 O-glycosylation in MUC2 (Figure 2.H).
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 .
B3GNT6 shows tissue-specific expression patterns that correlate with its functional significance in mucin biosynthesis:
Tissue Type | Relative Expression Level |
---|---|
Testis | High |
Rectum | High |
Duodenum | High |
Colon | High |
Appendix | High |
Other tissues | Variable/Lower |
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 .
B3GNT6 antibodies are utilized across multiple experimental techniques in glycobiology research:
Application | Common Dilutions | Key Considerations |
---|---|---|
Western Blot (WB) | 1:500-1:2000 | Expected band at ~43 kDa |
Immunohistochemistry (IHC) | 1:50-1:500 | Antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 |
Immunofluorescence (IF) | Variable (see specific product) | Primarily for subcellular localization studies |
ELISA | Variable (see specific product) | For quantitative detection |
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 .
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 .
Successful Western blot detection of B3GNT6 requires careful optimization:
Parameter | Recommendation | Rationale |
---|---|---|
Sample preparation | RIPA buffer extraction with freeze-thaw and syringe passage | Ensures efficient extraction of membrane-associated Golgi proteins |
Protein loading | 20-50 μg total protein | Sufficient for detection in most expressing tissues |
Blocking solution | 5% non-fat milk or BSA in TBST | Reduces non-specific binding |
Primary antibody dilution | Start with 1:1000, optimize between 1:500-1:2000 | Varies by antibody source and sample type |
Incubation conditions | Overnight at 4°C | Enhances specific binding |
Detection system | HRP-conjugated secondary with ECL or fluorescence-based detection | Choose based on sensitivity requirements |
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 .
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 .
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:
Challenge | Potential Causes | Solutions |
---|---|---|
High background in IHC | Insufficient blocking, excessive antibody concentration | Optimize blocking conditions (3-5% BSA or normal serum), titrate antibody, increase washing steps |
Weak or no signal in Western blot | Low expression, antibody sensitivity, inefficient extraction | Use tissues with known high expression, enrich target protein, optimize extraction method |
Multiple bands in Western blot | Isoforms, degradation, non-specific binding | Verify with knockout controls, use fresh samples with protease inhibitors, optimize blocking |
Inconsistent IHC staining | Fixation variability, antigen masking | Standardize fixation protocols, optimize antigen retrieval methods |
Variable results between experiments | Antibody lot variation, sample handling differences | Use consistent antibody lots, standardize sample processing |
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 .
Control Type | Recommendation | Application |
---|---|---|
Positive tissue controls | Mouse colon tissue, mouse small intestine tissue, mouse testis tissue | Western blot, IHC |
Positive cell line controls | Cell lines with known B3GNT6 expression (e.g., certain colorectal cancer lines) | Western blot, IF |
Negative controls | Primary antibody omission | All applications |
Specificity controls | CRISPR/Cas9 B3GNT6 knockdown/knockout samples | All applications |
Isotype controls | Matched isotype IgG at equivalent concentration | IHC, IF, Flow cytometry |
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 .
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 .
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:
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 .