mgat4c Antibody

Basic Research Considerations

Proper antibody validation requires multiple controls:

• Positive tissue controls: Mouse and rat testis tissues have been documented as positive controls for WB and IP applications .

• Knockout/knockdown validation: KD/KO testing has been documented in publications, providing the gold standard for antibody specificity .

• Antigen pre-absorption: Competition with the immunizing peptide can confirm binding specificity.

• Western blot molecular weight verification: MGAT4C should appear at approximately 55 kDa, consistent with its calculated molecular weight of 56 kDa .

• Cross-reactivity assessment: Test against related MGAT family members, particularly MGAT4A and MGAT4B, which share structural similarities .

How should sample preparation be optimized for MGAT4C detection in Western blotting?

Effective sample preparation for MGAT4C detection requires special consideration for this glycosyltransferase:

• Subcellular fractionation: As a Golgi-resident enzyme, enriching for membrane fractions can improve detection sensitivity.

• Lysis buffer composition: Use buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40) supplemented with protease inhibitors to preserve protein integrity.

• Glycoprotein considerations: MGAT4C itself is N-glycosylated, which may affect antibody recognition and migration patterns on SDS-PAGE . Consider including both native and PNGase F-treated samples to assess glycosylation-dependent antibody recognition.

• Reducing conditions: Ensure complete denaturation with standard SDS sample buffer containing β-mercaptoethanol or DTT.

• Sample concentration: For tissues with low expression, immunoprecipitation before Western blotting can improve detection. Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

What are the key considerations for immunohistochemical detection of MGAT4C?

For successful IHC applications with MGAT4C antibodies:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically most effective for exposing MGAT4C epitopes in formalin-fixed, paraffin-embedded tissues.

• Fixation sensitivity: Both commercial antibodies from Sigma-Aldrich and other vendors have been validated in formalin-fixed, paraffin-embedded tissue sections .

• Detection systems: Amplification methods such as polymer-based detection systems may improve sensitivity for this potentially low-abundance target.

• Subcellular localization: Expect predominantly perinuclear Golgi staining pattern consistent with MGAT4C's function in N-glycan processing.

• Tissue expression: Expression has been documented primarily in heart, adrenal gland, testis, liver, brain and fetal brain, with absent expression in pancreas .

How can MGAT4C antibodies be applied to investigate N-glycosylation mechanisms in cellular models?

MGAT4C antibodies can be strategically employed in several advanced experimental approaches:

• Co-immunoprecipitation studies: MGAT4C antibodies can be used to identify protein interactions, particularly with substrates like CD133 which has been identified as a direct substrate of MGAT4C .

• Glycan branching analysis: Combine MGAT4C immunoprecipitation with lectin blotting to correlate enzyme levels with N-glycan branching patterns.

• Structure-function studies: Recent research has revealed that MGAT4C, like other MGAT4 family members, contains both a catalytic domain and a lectin domain, the latter being essential for activity with glycoprotein substrates but not free glycans . Antibodies targeting different domains can help dissect these functional differences.

• Cellular trafficking: Immunofluorescence using MGAT4C antibodies can track Golgi organization and glycosylation enzyme localization during cellular stress or disease states.

• Combined with glycome analysis: Correlate MGAT4C protein expression levels with mass spectrometry-based glycan profiling to establish direct relationships between enzyme abundance and specific N-glycan structures.

What experimental approaches can differentiate between MGAT4C and related MGAT4 family members?

Distinguishing between MGAT4 family isozymes requires careful experimental design:

• Epitope selection: The antibody immunogens should target unique regions that don't overlap with other MGAT4 family members. Several commercial antibodies use the C-terminal region (e.g., YKGTENKLKDDDFEEESFDIPDNPPASLYTNMNVFENYEASKAYSSVDEYFWGKPPSTGDVFVIVFENPIIIKKIKVNTGTEDRQNDILHHGALDVGENVMPSKQRRQCSTYLRLGEFKNGNFEMSGVNQKIPFDIHCMRIYVTKTQ) as immunogen .

• Expression patterns: MGAT4C shows different tissue distribution compared to MGAT4A and MGAT4B, with strong expression in testis tissues .

• Functional assays: MGAT4C, unlike MGAT4A and MGAT4B, does not catalyze GlcNAc transfer to the Manα1-6 arm to form GlcNAcβ1-4Manα1-6 linkage ('GnT-VI' activity) .

• Substrate specificity: Research indicates that MGAT4C is involved in processing complex N-glycan structures specifically on CD133, making this a useful differential marker .

• Genetic approaches: Use of CRISPR-engineered knockout cell lines for each MGAT family member can provide definitive controls for antibody specificity testing.

How can MGAT4C antibodies be utilized to investigate glycosylation changes in pathological conditions?

MGAT4C antibodies can facilitate several research strategies in disease contexts:

• Expression analysis in tissue microarrays: Compare MGAT4C levels across normal and pathological tissues to identify disease-associated alterations in glycosylation pathways.

• Cancer stem cell research: Given MGAT4C's involvement in CD133 glycosylation and CD133's role as a marker for numerous stem cells and cancer stem cells, antibodies can help investigate the relationship between glycosylation and cancer stemness .

• Neurodevelopmental disorders: Abnormal N-acetylglucosaminyltransferase expression in prefrontal cortex has been linked to schizophrenia, suggesting potential applications in neuropsychiatric research .

• Aberrant glycosylation profiling: MGAT4C antibodies can identify altered expression in diseases characterized by glycosylation defects, potentially revealing new biomarkers.

• Therapeutic development: In combination with functional studies, antibody-based detection of MGAT4C could identify contexts where modulation of tri-antennary N-glycan formation might have therapeutic value.

What approaches can resolve conflicting MGAT4C expression data between different antibodies?

When facing inconsistent results between different MGAT4C antibodies:

• Epitope mapping: Determine whether antibodies recognize different domains of MGAT4C. Recent research shows MGAT4C contains both a catalytic domain and a lectin domain with distinct functions .

• Glycosylation-dependent recognition: Test whether antibody binding is affected by the glycosylation state of MGAT4C itself, as research shows MGAT4C's own N-glycans contain self-ligands for its lectin domain .

• Cross-validation approaches: Employ multiple detection methods (e.g., mass spectrometry, RT-PCR) to confirm protein and transcript levels.

• Isoform specificity: Determine whether discrepancies arise from detection of different splice variants or post-translationally modified forms.

• Knockout validation: Generate CRISPR knockout controls in relevant cell types to definitively establish antibody specificity.

How can MGAT4C antibodies contribute to glycoengineering of therapeutic antibodies?

MGAT4C antibodies can support several aspects of glycoengineering research:

• Expression monitoring: Track MGAT4C expression levels in engineered cell lines used for therapeutic antibody production.

• Pathway verification: Confirm successful manipulation of N-glycan branching pathways in CHO cells or other production systems. Recent research has employed multiplexed engineering of glycosyltransferase genes including MGAT5 to produce antibodies with tri-antennary N-glycans .

• Structure-function correlations: Investigate how MGAT4C contributes to specific N-glycan structures that affect therapeutic antibody properties.

• Quality control: Monitor glycosylation enzyme expression during manufacturing to ensure consistent glycoform profiles.

• Combinatorial approach: Research has shown that combinatorial expression of multiple glycosyltransferases (B4GalT1, ST6Gal1, and MGAT family enzymes) can produce antibodies with designer glycan structures with potential therapeutic advantages .

What advanced methodologies can determine MGAT4C's contribution to specific N-glycan structures?

To precisely define MGAT4C's glycan synthesis role:

• CRISPR-based genetic manipulation: Generate MGAT4C knockout, knockdown, and overexpression models followed by comprehensive glycan analysis.

• Site-specific glycoprofiling: Combine immunoprecipitation, enzymatic digestion, and mass spectrometry to identify MGAT4C-dependent glycosylation sites on target proteins.

• In vitro enzymatic assays: Use purified MGAT4C and defined substrates to characterize enzymatic properties and substrate preferences.

• Structural biology approaches: Antibodies can aid crystallography studies by stabilizing MGAT4C conformations, revealing structure-function relationships as has been done with related glycosyltransferases.

• Lectin domain characterization: Recent research reveals MGAT4C contains a lectin domain essential for activity toward glycoprotein substrates but dispensable for free glycan modification, suggesting complex regulatory mechanisms .

How should researchers address non-specific binding with MGAT4C antibodies?

When encountering non-specific bands in MGAT4C detection:

• Optimization of blocking conditions: Test alternative blocking agents (5% non-fat milk, 5% BSA, commercial blocking buffers) to reduce background.

• Titration of antibody concentration: Perform a dilution series (1:500 to 1:2000 for WB) to determine optimal signal-to-noise ratio .

• Pre-adsorption strategy: Pre-adsorb antibody with the immunizing peptide when available to verify the specificity of bands.

• Protein extraction method: Optimize extraction protocols to reduce co-purification of proteins with similar molecular weights.

• Alternative antibody validation: When possible, test multiple antibodies targeting different epitopes of MGAT4C to confirm specific bands. This is particularly important given the limited validation data for some commercial MGAT4C antibodies.

What are the critical considerations for reproducible immunoprecipitation with MGAT4C antibodies?

For successful MGAT4C immunoprecipitation:

• Antibody quantity optimization: Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate, as recommended in validated protocols .

• Lysis buffer selection: Use non-denaturing buffers containing 1% NP-40 or Triton X-100 to preserve native protein structure while effectively solubilizing membrane-associated MGAT4C.

• Cross-linking consideration: For capturing transient interactions, consider using membrane-permeable cross-linkers before cell lysis.

• Wash stringency balance: Optimize wash conditions to remove non-specific interactions while retaining genuine MGAT4C complexes.

• Validated positive controls: Include mouse testis tissue as a positive control, which has been documented to contain detectable MGAT4C levels .