MGAT4C Antibody

What is MGAT4C and what is its biological function?

MGAT4C (mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme C) is a glycosyltransferase that catalyzes the formation of the GlcNAcbeta1-4 branch on the GlcNAcbeta1-2Manalpha1-3 arm of N-linked glycans. It plays an essential role in producing tri- and tetra-antennary N-linked sugar chains . MGAT4C is part of the GnT-IV family, which includes four homologous members in humans: GnT-IVa (MGAT4A), GnT-IVb (MGAT4B), GnT-IVc (MGAT4C, also known as GnT-VI), and GnT-IVd (MGAT4D, also known as GnT-1IP) . Functionally, MGAT4C participates in N-glycan processing of certain proteins, with CD133 identified as one of its substrates, affecting cell surface recognition patterns .

What types of MGAT4C antibodies are available for research?

MGAT4C antibodies are primarily available as polyclonal antibodies derived from rabbits. The most commonly used ones in research include:

Most commercially available antibodies are purified through protein A columns followed by peptide affinity purification, and are typically supplied in PBS with preservatives like sodium azide .

What are the validated applications for MGAT4C antibodies?

MGAT4C antibodies have been validated for several research applications:

• Western Blot (WB): Most MGAT4C antibodies are validated for WB with recommended dilutions ranging from 1:500 to 1:2000 . For example, MGAT4C antibody from Proteintech has been successfully used to detect the protein at approximately 55 kDa in HepG2 cell line lysates .

• Immunohistochemistry (IHC): MGAT4C antibodies have been validated for IHC in formalin-fixed paraffin-embedded tissues, particularly in human skeletal muscle samples . Typical dilutions range from 1:10 to 1:50.

• Immunoprecipitation (IP): Some antibodies, like the one from Proteintech, have been validated for IP using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• ELISA: Most MGAT4C antibodies can be used in ELISA applications, though specific protocols may vary by manufacturer .

How should I design an experiment to study MGAT4C's role in glycosylation using these antibodies?

When studying MGAT4C's role in glycosylation processes, consider the following experimental design approach:

• Cell/Tissue Selection: Choose cell lines or tissues known to express MGAT4C. Studies have identified testis tissue (mouse and rat) as good sources for MGAT4C detection .

• Expression Analysis:

  • Use Western blot to confirm MGAT4C expression levels

  • Complement with qRT-PCR to correlate protein and mRNA levels

• Functional Studies:

  • Design UDP-Glo enzyme assays to measure MGAT4C activity toward glycoprotein substrates

  • Use N-glycan structural analysis through techniques like mass spectrometry to evaluate branching patterns

• Substrate Interaction:

  • For studying CD133 as a substrate, use co-immunoprecipitation with your MGAT4C antibody

  • Consider RNAi experiments targeting MGAT4C to observe effects on CD133 glycosylation and cell surface expression

• Controls:

  • Use MGAT4A and MGAT4B as comparative controls since they have distinct glycoprotein preferences

  • Include glycosylation inhibitors like tunicamycin as experimental controls

What are the optimal conditions for storing and handling MGAT4C antibodies?

For optimal preservation of MGAT4C antibody activity:

Why might I be experiencing high background or non-specific binding with my MGAT4C antibody?

High background or non-specific binding can occur for several reasons when using MGAT4C antibodies:

• Antibody Concentration: Using too high a concentration can lead to non-specific binding. Start with the manufacturer's recommended dilution and optimize as needed .

• Blocking Efficiency: Insufficient blocking can lead to high background. Ensure adequate blocking with 5% BSA or milk in TBST/PBST.

• Cross-Reactivity: Polyclonal antibodies may cross-react with structurally similar proteins, particularly other MGAT family members. Consider pre-absorption with related proteins or using more stringent washing conditions.

• Sample Preparation: Improper fixation or permeabilization can affect epitope accessibility and increase non-specific binding.

• Quality of Serum: Serum quality matters significantly. Poor quality serum with high hemoglobin concentration (>1 g/l) can affect immune staining by increasing fluorescent or HRP-reactive background .

• Solution: Use proper controls (pre-immune serum from the same animal as a negative control), optimize antibody concentration, increase wash stringency, and consider using a more specific detection system.

How can I effectively use MGAT4C antibodies to study the relationship between glycosylation and disease states?

To investigate MGAT4C's role in disease-related glycosylation changes:

• Tissue Microarray Analysis: Use MGAT4C antibodies for IHC on tissue microarrays containing normal and pathological samples to identify differential expression patterns.

• Cell Models:

  • Create MGAT4C knockout or overexpression cell lines to study glycosylation effects

  • Use UDP-Glo assays to compare enzymatic activity between normal and disease models

• Glycoprotein Target Identification:

  • Employ co-immunoprecipitation with MGAT4C antibodies followed by mass spectrometry to identify novel substrates

  • Confirm substrate relationships with specific glycoproteins (like CD133)

• Sequential Immunoprecipitation:

  • Perform sequential IP with MGAT4C antibody and then substrate-specific antibodies to investigate complex formation

  • Analyze glycan structures of precipitated complexes using lectins or glycan-specific antibodies

• Comparative Studies:

  • Compare MGAT4C expression/activity with that of MGAT4A and MGAT4B, as these isozymes have demonstrated distinct glycoprotein preferences

  • Correlate findings with disease progression or biomarker potential

What strategies can I use to validate MGAT4C antibody specificity for my particular research application?

Validating MGAT4C antibody specificity is crucial for reliable research outcomes:

• Knockout/Knockdown Controls:

  • Use CRISPR/Cas9 or RNAi to generate MGAT4C-deficient cells

  • Confirm antibody specificity by demonstrating reduced or absent signal in these models

• Overexpression Validation:

  • Transfect cells with MGAT4C expression vectors to create positive controls

  • Verify increased signal intensity correlating with overexpression levels

• Peptide Competition Assays:

  • Pre-incubate the antibody with its immunizing peptide

  • Confirm signal reduction in subsequent applications

• Cross-reactivity Assessment:

  • Test the antibody against recombinant MGAT4A and MGAT4B proteins

  • Ensure sufficient distinction between these closely related family members

• Multi-antibody Verification:

  • Use multiple antibodies targeting different epitopes of MGAT4C

  • Confirm consistent localization/detection patterns

• Technical Controls:

  • Include isotype controls (for monoclonal) or pre-immune serum (for polyclonal)

  • Perform parallel experiments with secondary antibodies alone

How can single-cell analysis techniques be combined with MGAT4C antibodies to advance glycobiology research?

Integrating MGAT4C antibodies with single-cell techniques offers powerful new research approaches:

• Single-Cell Antibody Sequencing:

  • Adapt the methods described for B cell antibody sequencing to analyze cellular responses to MGAT4C-related glycan modifications

  • Combine FACS sorting of individual cells with targeted RT-PCR for MGAT4C and related genes

• Flow Cytometry Applications:

  • Develop intracellular staining protocols for MGAT4C combined with surface glycan markers

  • Analyze heterogeneity in glycosylation patterns at the single-cell level

• Single-Cell RNA-Seq Integration:

  • Correlate MGAT4C protein levels (detected by antibody-based methods) with transcriptomic profiles

  • Identify co-expression networks related to glycan processing

• Spatial Transcriptomics:

  • Combine MGAT4C antibody staining with spatial transcriptomics to map glycosylation enzyme distribution in tissues

  • Relate spatial patterns to functional microenvironments

• Customized Workflows:

  • Develop flexible, customizable suites of methods from flow cytometry to single-cell sorting, similar to approaches used in antibody sequencing

  • Adapt these methods specifically for glycosyltransferase research

What are the considerations for using MGAT4C antibodies in bispecific antibody development and therapeutic research?

While primarily a research tool, MGAT4C antibodies have potential applications in therapeutic research:

• Bispecific Modeling Framework:

  • Consider using modeling frameworks similar to those described for other therapeutic antibodies

  • Predict efficacy, toxicity, and therapeutic index for any potential MGAT4C-targeting therapeutics

• Target Validation:

  • Use MGAT4C antibodies to validate this enzyme as a potential therapeutic target in disease models

  • Confirm specific glycosylation changes through functional inhibition studies

• Epitope Selection:

  • Carefully select epitopes that will not cross-react with other MGAT family members

  • Consider using structure-based design to target catalytic domains specifically

• Pharmacokinetic Considerations:

  • Factor in the subcellular localization of MGAT4C (primarily Golgi-associated)

  • Design antibody-based therapeutics that can access intracellular compartments if needed

• Predictive Modeling:

  • Employ in vitro receptor occupancy and pharmacokinetics data to guide therapeutic antibody development

  • Estimate required target occupancy thresholds for efficacy versus toxicity

What are the recommended protocols for optimizing MGAT4C antibody performance in challenging tissue samples?

When working with challenging samples, consider these optimization strategies:

• Antigen Retrieval Optimization:

  • For IHC applications, systematically test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

• Fixation Considerations:

  • Test multiple fixation protocols to determine optimal epitope preservation

  • Compare results from freshly frozen vs. FFPE tissues to determine epitope sensitivity to formalin

• Signal Amplification:

  • Implement tyramide signal amplification for low-abundance targets

  • Consider using polymer-based detection systems for enhanced sensitivity

• Background Reduction:

  • Incorporate additional blocking steps with normal serum from the secondary antibody host species

  • Include avidin/biotin blocking when using biotin-based detection systems

• Tissue-Specific Protocols:

  • For muscle tissue (shown to express MGAT4C ), use specialized permeabilization approaches

  • For testis tissue (where MGAT4C has been detected ), optimize antigen retrieval time

How can I design experiments to investigate the functional relationship between MGAT4C and its lectin domain in glycoprotein recognition?

Research suggests that the lectin domain in MGAT4 family proteins is critical for activity. To investigate MGAT4C's lectin domain:

• Domain Mutation Studies:

  • Generate MGAT4C constructs with mutations in the lectin domain

  • Compare enzymatic activity toward glycoprotein substrates vs. free glycans

• UDP-Glo Enzyme Assays:

  • Implement UDP-Glo assays to measure activity levels of wild-type vs. lectin domain mutants

  • Test activity on multiple glycoprotein substrates such as alpha-1-antitrypsin, haptoglobin, and alpha-1-acid glycoprotein

• Structural Analysis:

  • Use antibodies targeting specific epitopes to investigate conformational changes

  • Combine with biophysical techniques to understand domain interactions

• Glycoprotein Preference Profiling:

  • Similar to studies done with MGAT4A/B , characterize MGAT4C's glycoprotein preferences

  • Determine if MGAT4C shows distinct substrate specificity patterns compared to other family members

• Self-Regulation Studies:

  • Investigate whether MGAT4C exhibits self-regulation of activity through its own N-glycan modifications

  • Design experiments to determine if MGAT4C's lectin domain recognizes its own product N-glycan branches

By addressing these questions methodically, researchers can gain deeper insights into MGAT4C's function and potential applications in glycobiology research and beyond.