B4GALT4 Antibody

What is B4GALT4 and what are its key biological functions?

B4GALT4 is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes that encode type II membrane-bound glycoproteins. These enzymes show specificity for the donor substrate UDP-galactose and transfer galactose in a beta1,4 linkage to acceptor sugars including GlcNAc, Glc, and Xyl . B4GALT4 primarily functions in glycolipid biosynthesis and is involved in the synthesis of terminal N-acetyllactosamine (LacNac) units present on glycan chains of glycoproteins and glycosphingolipids . It catalyzes the transfer of galactose residues via a beta1->4 linkage from UDP-Gal to the non-reducing terminal N-acetyl glucosamine 6-O-sulfate in keratan sulfate proteoglycans . Additionally, B4GALT4 contributes to the generation of sialyl-Lewis X (sLex) epitopes on mucin-type glycoproteins that serve as ligands for L-selectin, a major regulator of leukocyte migration .

What types of B4GALT4 antibodies are commercially available?

Several types of B4GALT4 antibodies are commercially available with varying characteristics:

These antibodies are generated using various immunogens including recombinant fusion proteins and peptide sequences corresponding to different regions of human B4GALT4 .

What are the recommended protocols for Western blot using B4GALT4 antibodies?

For optimal Western blot results with B4GALT4 antibodies, follow these methodological guidelines:

• Sample Preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Load 20-50 μg of total protein per lane (varies by sample type)

• Antibody Dilutions:

  • Primary antibody: 1:1000-1:6000 (e.g., Proteintech 30478-1-AP) or 1:1000-1:4000 (e.g., Proteintech 15200-1-AP)

  • Secondary antibody: 1:2500-1:5000 HRP-conjugated appropriate secondary

• Positive Controls:

  • Cell lines: HT-29, A375, HEK293T, Caco-2, PC-3, HaCaT, HepG2

  • Tissues: Mouse kidney, lung, thymus, cerebellum; rat kidney and thymus

• Expected Results:

  • Primary band at 34-42 kDa

  • Multiple bands may indicate different glycosylation states or splice variants

• Optimization Tips:

  • Perform antibody titration to determine optimal concentration

  • Include positive controls from validated samples

  • If necessary, extend blocking time to reduce background

What is the recommended protocol for immunofluorescence using B4GALT4 antibodies?

For immunofluorescence applications with B4GALT4 antibodies:

• Cell Preparation:

  • Grow cells on coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

• Antibody Dilutions:

  • Primary antibody: 1:50-1:500 (e.g., Proteintech 15200-1-AP) or 1:1000-1:4000 (e.g., Proteintech 30478-1-AP)

  • Secondary antibody: 1:500-1:1000 fluorophore-conjugated appropriate secondary

• Validated Cell Lines:

  • A549 cells

  • HeLa cells

• Expected Staining Pattern:

  • Perinuclear Golgi apparatus localization (consistent with type II membrane protein localization in Golgi)

• Controls:

  • Include secondary-only controls

  • Consider co-staining with Golgi markers for colocalization analysis

What methods should be used for immunohistochemistry with B4GALT4 antibodies?

For immunohistochemistry applications:

• Tissue Preparation:

  • FFPE tissue sections (4-6 μm thickness)

  • Deparaffinize and rehydrate through graded alcohols

• Antigen Retrieval:

  • Use TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 (alternative)

  • Heat-induced epitope retrieval: 95-98°C for 15-20 minutes

• Antibody Dilutions:

  • Primary antibody: 1:50-1:500 (e.g., Proteintech 15200-1-AP)

  • Secondary detection: Use appropriate HRP-polymer system

• Validated Tissues:

  • Human kidney tissue has been validated for IHC

• Controls and Optimization:

  • Include isotype controls

  • Use positive control tissues (human kidney)

  • Optimize antibody concentration and incubation time based on signal intensity and background

How can I validate the specificity of B4GALT4 antibodies?

To ensure antibody specificity, implement these validation strategies:

• Positive and Negative Controls:

  • Positive controls: Use cell lines with confirmed B4GALT4 expression such as 293T, A431, Jurkat, and Raji

  • Negative controls: Use B4GALT4-deficient cell lines generated by CRISPR methods as described in Kizuka et al.

• Multiple Antibody Approach:

  • Test multiple antibodies targeting different regions of B4GALT4

  • Compare staining patterns across antibodies targeting different epitopes (e.g., antibodies against AA 1-344, AA 35-134, AA 91-117)

• Blocking Peptide Experiments:

  • Pre-incubate antibody with immunizing peptide or recombinant protein

  • Monitor elimination of specific signal

• Genetic Validation:

  • Use siRNA knockdown or CRISPR knockout systems

  • Confirm reduction or absence of B4GALT4 signal

• Orthogonal Techniques:

  • Correlate antibody results with mRNA expression data

  • Validate with mass spectrometry if possible

What controls should be included when working with B4GALT4 antibodies?

Always include these controls in experiments with B4GALT4 antibodies:

• Positive Controls:

  • Cell lines: HT-29, A375, HEK293T, Caco-2, PC-3, HaCaT, HepG2

  • Tissues: Mouse kidney, lung, thymus; rat kidney and thymus; human kidney

• Negative Controls:

  • Primary antibody omission

  • Isotype-matched non-specific antibodies

  • B4GALT4 knockout or knockdown samples when available

• Technical Controls:

  • Antibody titration controls

  • Secondary antibody-only controls

  • Blocking peptide controls

• Biological Relevance Controls:

  • Compare expression across multiple cell types

  • Include developmentally or physiologically relevant controls

How should experimental conditions be optimized for B4GALT4 antibody specificity?

For optimal B4GALT4 antibody performance, consider these experimental optimizations:

• Sample Preparation:

  • For membrane proteins like B4GALT4, use detergent-based extraction methods

  • Include protease and phosphatase inhibitors in lysis buffers

  • For IHC/IF, optimize fixation method and duration

• Blocking Conditions:

  • Test different blocking agents (BSA, normal serum, milk)

  • Optimize blocking time and temperature

  • Use blocking agent compatible with detection system

• Antibody Dilution and Incubation:

  • Perform titration experiments (1:500-1:6000 for most applications)

  • Test different incubation times and temperatures

  • Consider overnight incubation at 4°C for maximum sensitivity

• Washing Procedures:

  • Optimize wash buffer composition (TBST or PBST)

  • Increase wash duration or frequency to reduce background

  • Ensure complete removal of wash buffer between steps

• Detection System Optimization:

  • Select appropriate secondary antibody host species

  • Optimize signal amplification methods if needed

  • Adjust exposure times for optimal signal-to-noise ratio

Why might I observe multiple bands in Western blot using B4GALT4 antibodies?

Multiple bands in B4GALT4 Western blots can result from several factors:

• Glycosylation Heterogeneity:
  B4GALT4 contains N-glycosylation sites, including Asn220, which is crucial for enzymatic activity . Different glycosylation states can produce proteins with varying molecular weights.

• Splice Variants:
  Two alternatively spliced transcript variants have been identified for the B4GALT4 gene , potentially resulting in protein isoforms of different sizes.

• Proteolytic Processing:
  As a type II membrane protein, B4GALT4 may undergo proteolytic processing during sample preparation or naturally within cells.

• Cross-reactivity with Related Proteins:
  B4GALT4 belongs to a family of seven beta-1,4-galactosyltransferases with sequence similarities. By sequence homology, B4GALT3 and B4GALT4 form a subgroup , potentially leading to cross-reactivity.

• Experimental Artifacts:

  • Incomplete denaturation

  • Protein aggregation

  • Sample degradation

  • Non-specific antibody binding

To distinguish between these possibilities, consider:

• Deglycosylation experiments using PNGase F

• Testing multiple antibodies targeting different epitopes

• Including B4GALT4 knockout controls

• Optimizing sample preparation conditions

How can I address weak or absent signal when using B4GALT4 antibodies?

If experiencing weak or no signal with B4GALT4 antibodies, systematically address these potential causes:

• Antibody-Related Issues:

  • Verify antibody concentration (try more concentrated solution)

  • Check antibody storage conditions and expiration date

  • Consider a different antibody targeting an alternative epitope

• Sample-Related Issues:

  • Confirm B4GALT4 expression in your sample type

  • Use positive controls known to express B4GALT4 (e.g., HT-29 cells)

  • Optimize protein extraction method for membrane proteins

  • Increase protein loading amount

• Protocol Optimization:

  • Enhance antigen retrieval (for IHC/IF)

  • Increase antibody incubation time

  • Optimize blocking conditions

  • Enhance detection sensitivity with signal amplification

• Technical Considerations:

  • Verify secondary antibody compatibility

  • Check detection reagents (fresh ECL solution for WB)

  • Ensure equipment is functioning properly (microscope settings, imager exposure)

• Biological Considerations:

  • B4GALT4 may be expressed at low levels in your sample

  • Post-translational modifications may mask epitopes

  • Consider if experimental conditions might downregulate B4GALT4

How does glycosylation affect B4GALT4 detection with antibodies?

B4GALT4 glycosylation significantly impacts antibody detection in several ways:

• Critical Functional Role:
  N-glycosylation of B4GALT4 is crucial for its enzymatic activity, with two identified N-glycosylation sites, particularly Asn220 having substantial impact on function .

• Molecular Weight Variation:
  Glycosylation contributes to the observed molecular weight variations (34-42 kDa) despite a calculated weight of 40 kDa .

• Epitope Accessibility:
  Glycan structures may mask epitopes, particularly if the antibody recognition site is near or includes a glycosylation site.

• Heterogeneous Detection:
  Different glycoforms may be detected with varying efficiency, potentially resulting in multiple bands or variable signal intensity.

• Cell Type Variations:
  Different cell types may express B4GALT4 with distinct glycosylation patterns, affecting detection consistency across samples.

Methodological approaches to address glycosylation effects include:

• Deglycosylation experiments to confirm glycosylation-dependent changes

• Using multiple antibodies targeting different epitopes

• Comparing detection patterns across different cell types and tissues

• Correlating antibody detection with functional activity assays

How can B4GALT4 antibodies facilitate glycosylation pathway research?

B4GALT4 antibodies serve as valuable tools for investigating glycosylation pathways through these methodological approaches:

• Localization and Co-localization Studies:

  • Use immunofluorescence to visualize B4GALT4 localization in the Golgi apparatus

  • Perform co-localization studies with other glycosyltransferases to map spatial organization of glycosylation machinery

  • Correlate with lectin staining to connect enzyme localization with specific glycan structures

• Protein Complex Identification:

  • Immunoprecipitate B4GALT4 to identify interaction partners

  • Investigate complexes with B3GNT7 and sulfotransferases CHST6 and CHST1, which cooperate in keratan sulfate synthesis

  • Perform proximity ligation assays to visualize protein-protein interactions in situ

• Expression Analysis in Disease Models:

  • Study B4GALT4 expression in Congenital Disorders of Glycosylation

  • Investigate expression changes in cancer models and correlate with altered glycosylation patterns

  • Examine regulation in immune disorders where selectin ligands play important roles

• Functional Studies with Genetic Manipulation:

  • Use antibodies to validate knockdown/knockout models

  • Correlate B4GALT4 expression with glycan profile changes using mass spectrometry

  • Perform rescue experiments with wild-type vs. mutant B4GALT4

• Structure-Function Relationships:

  • Use antibodies recognizing different domains to study protein topology

  • Correlate domain-specific antibody binding with enzymatic activity

  • Investigate effects of mutations on protein expression and localization

What role does B4GALT4 play in disease models and how can antibodies help investigate this?

B4GALT4 is implicated in several disease contexts that can be investigated using antibodies:

• Congenital Disorders of Glycosylation:

  • B4GALT4 is associated with Congenital Disorder of Glycosylation, Type IId

  • Antibodies can assess expression levels and localization in patient-derived samples

  • Comparative studies between normal and pathological tissues can reveal disease mechanisms

• Cancer Biology:

  • Altered glycosylation is a hallmark of cancer

  • B4GALT4 contributes to synthesis of sialyl-Lewis X, which plays roles in metastasis

  • Antibodies can track B4GALT4 expression changes during cancer progression

• Inflammatory and Immune Disorders:

  • B4GALT4 contributes to L-selectin ligand synthesis, important for leukocyte migration

  • Antibodies can examine B4GALT4 regulation during inflammatory responses

  • Co-localization studies with inflammation markers can reveal regulatory mechanisms

• Experimental Approaches:

  • IHC on tissue microarrays to assess expression across disease stages

  • Correlation of B4GALT4 levels with specific glycan profiles in disease tissues

  • Manipulation of B4GALT4 expression in disease models to assess functional outcomes

• Potential Therapeutic Applications:

  • Use antibodies to validate B4GALT4 as a potential therapeutic target

  • Develop assays to screen for B4GALT4 modulators

  • Monitor B4GALT4 expression changes in response to treatments

How can B4GALT4 antibodies be integrated with other techniques for comprehensive glycobiology research?

Integrating B4GALT4 antibodies with complementary techniques creates powerful research approaches:

• Antibody-Glycan Staining Correlations:

  • Combine B4GALT4 immunostaining with lectin histochemistry

  • Correlate enzyme expression with specific glycan structures

  • Map spatial relationships between enzyme localization and product glycans

• Multi-omics Integration:

  • Link B4GALT4 protein expression (antibody-based detection) with:

    • Transcriptomics (RNA-seq for expression regulation)

    • Glycomics (mass spectrometry for glycan profiling)

    • Interactomics (IP-MS for protein interaction networks)

• CRISPR-Based Functional Studies:

  • Generate B4GALT4 knockout models using CRISPR as described by Kizuka et al.

  • Verify knockout with antibodies

  • Characterize glycan alterations using mass spectrometry

  • Perform rescue experiments with wild-type or mutant B4GALT4

• Subcellular Fractionation Analysis:

  • Use B4GALT4 antibodies to track enzyme distribution during subcellular fractionation

  • Analyze glycosyltransferase activity in different cellular compartments

  • Correlate enzyme localization with functional activity

• Live-Cell Dynamics:

  • Combine antibody data with live-cell imaging of fluorescently tagged B4GALT4

  • Study trafficking and localization dynamics in response to cellular stimuli

  • Investigate protein turnover and regulation mechanisms

• Therapeutic Development Applications:

  • Use antibodies to validate target engagement in drug discovery

  • Develop screening assays for modulators of B4GALT4 expression or activity

  • Monitor treatment effects on B4GALT4 expression and function