MS4A8 Antibody

What is MS4A8 and why is it significant in biological research?

MS4A8 (Membrane-Spanning 4-Domains, Subfamily A, Member 8) is a multi-pass membrane protein belonging to the MS4A family. Also known as MS4A8B, 4SPAN4, or CD20L5, it is located on chromosome 11q12.2 in humans . This protein is primarily expressed in hematopoietic tissues and cell lines, and is suspected to function in signal transduction as a component of a multimeric receptor complex .

The significance of MS4A8 in research has increased due to recent findings linking the MS4A gene cluster to Alzheimer's disease pathogenesis. Specifically, variants in the MS4A locus modify risk for late-onset Alzheimer disease and modulate extracellular soluble TREM2 (Triggering Receptor Expressed on Myeloid cells 2), an important molecule in neuroinflammatory processes . Understanding MS4A8's biological functions through antibody-based detection methods has therefore become increasingly important in neurological and immunological research.

How do polyclonal and monoclonal MS4A8 antibodies differ in research applications?

Polyclonal and monoclonal MS4A8 antibodies offer distinct advantages for different research applications:

What epitopes of MS4A8 are commonly targeted by research-grade antibodies?

Commercial MS4A8 antibodies target several distinct epitopes, with predominant focus on the N-terminal region:

• N-terminal region (amino acids 44-70): Multiple vendors offer antibodies targeting this region, which appears to be immunogenic and accessible .

• Amino acids 20-74: This slightly larger N-terminal region is targeted by several antibodies available with different conjugations (FITC, biotin, HRP) .

• Amino acids 100-200: Some antibodies target this internal region of the human MS4A8B protein (NP_113645.1) .

• Full-length (AA 1-250): Antibodies targeting the complete protein are also available for applications requiring recognition of the intact protein .

When selecting antibodies based on epitope, consider the protein's topology as a multi-pass membrane protein. The N-terminal region (AA 44-70) appears to be a common choice for detection, likely due to its accessibility and specificity .

What applications are best supported by validated MS4A8 antibodies?

Based on available validation data, MS4A8 antibodies support several key applications with varying levels of validation evidence:

Western blotting represents the most consistently validated application across products, with documented detection of MS4A8 at its expected molecular weight of approximately 26 kDa . When selecting antibodies for less commonly validated applications such as IHC or IF, researchers should prioritize products with application-specific validation data and consider performing preliminary validation experiments .

How should sample preparation be optimized for MS4A8 detection in different experimental contexts?

Sample preparation significantly impacts MS4A8 detection success across different experimental methods:

For Western Blotting:

• Extract proteins using buffers containing detergents suitable for membrane proteins (e.g., RIPA buffer with 0.1% SDS)

• Use fresh samples where possible, as MS4A8 stability in long-term storage has not been well-characterized

• Recommended dilutions range from 1:500-1:2,000 for most commercial antibodies

• U-87MG cells have been validated as a positive control for some antibodies

For Immunohistochemistry:

• Antigen retrieval methods vary by antibody but typically involve heat-induced epitope retrieval in citrate buffer (pH 6.0)

• The Human Protein Atlas recommends specific antigen retrieval protocols for MS4A8 detection in fixed tissues

For Flow Cytometry and IF:

• When using FITC-conjugated MS4A8 antibodies, minimize exposure to light during preparation

• For membrane protein detection, gentle fixation (2-4% paraformaldehyde) and careful permeabilization are critical

• Storage buffer typically consists of PBS (pH 7.2-7.4) with 0.09% sodium azide for stability

In all applications, include appropriate blocking steps using either 5% BSA or 5% non-fat milk in TBS-T to minimize non-specific binding.

What controls should be incorporated when validating MS4A8 antibodies for new applications?

Comprehensive validation of MS4A8 antibodies requires multiple controls:

Positive Controls:

• Human hematopoietic tissues and cell lines with confirmed MS4A8 expression

• U-87MG cells have been validated as expressing MS4A8 and can serve as a positive control for Western blot

• Recombinant MS4A8 protein (where available) provides a defined positive control

Negative Controls:

• Tissues known not to express MS4A8 (the Human Protein Atlas provides expression data across tissues)

• Primary antibody omission controls to assess secondary antibody specificity

• Isotype controls: For monoclonal antibodies, use the corresponding mouse IgG (e.g., for clone 1B4)

• For polyclonal antibodies, rabbit IgG serves as an appropriate isotype control

Technical Validation Approaches:

• Antibody absorption test: Pre-incubate the antibody with immunizing peptide (when available) to confirm specificity

• RNAi validation: Use MS4A8-targeted siRNA/shRNA to create knockdown samples, which should show reduced signal compared to controls

• Multiple antibody approach: Use two antibodies targeting different MS4A8 epitopes to confirm detection specificity

For the most rigorous validation, consider orthogonal validation comparing antibody-based detection with MS4A8 mRNA quantification using qPCR.

How can MS4A8 antibodies be utilized to study the MS4A gene cluster's role in modulating TREM2 signaling?

Recent research has identified the MS4A gene cluster as a key modulator of soluble TREM2 (sTREM2) and Alzheimer's disease pathogenesis. MS4A8 antibodies can be employed in several advanced approaches to investigate this relationship:

• Co-immunoprecipitation studies: MS4A8 antibodies can be used to pull down protein complexes to identify physical interactions between MS4A8 and TREM2 or other members of the signaling pathway. Research has shown that MS4A4A (a related family member) colocalizes with TREM2 on lipid rafts at the plasma membrane and modulates sTREM2 levels .

• Proximity ligation assays: This technique can detect protein-protein interactions between MS4A8 and potential binding partners in the TREM2 pathway with spatial resolution in fixed cells or tissues.

• Immunofluorescence co-localization: MS4A8 antibodies can be paired with TREM2 antibodies in dual-labeling experiments to examine their spatial relationship, particularly in microglia, where these interactions are likely to occur.

• Genetic association studies: MS4A8 antibodies can be used to quantify protein levels in individuals with different MS4A locus variants (e.g., rs1582763) to correlate genotype with protein expression and sTREM2 levels .

When designing such experiments, it's critical to consider that the rs1582763 variant is associated with increased CSF sTREM2 and reduced AD risk, suggesting that MS4A gene products may modulate TREM2 processing or shedding . MS4A8 antibodies provide a tool to investigate whether MS4A8 shares the regulatory functions observed for MS4A4A in this pathway.

What approaches can be used to design antibodies with customized specificity profiles for MS4A family research?

Developing antibodies with customized specificity profiles for MS4A family research requires sophisticated approaches:

• Biophysics-informed modeling: Recent research has demonstrated the successful design of antibodies with customized specificity profiles using biophysics-informed models trained on experimentally selected antibodies. This approach associates distinct binding modes with each potential ligand, enabling prediction and generation of variants beyond those observed in experiments .

• Epitope selection strategies: When designing MS4A8-specific antibodies, targeting unique regions with low homology to other MS4A family members is crucial. Sequence analysis reveals that the N-terminal regions (e.g., AA 44-70) offer greater differentiation from other family members .

• Phage display optimization: Phage display experiments with antibody selection against diverse combinations of closely related ligands can help identify and disentangle multiple binding modes associated with specific members of the MS4A family .

• Cross-specificity engineering: For research requiring simultaneous detection of multiple MS4A family members, antibodies can be engineered for controlled cross-reactivity by targeting conserved epitopes, while strict specificity can be achieved by targeting unique regions.

When using this approach, researchers should perform rigorous validation using both positive controls (MS4A8-expressing samples) and negative controls (samples expressing other MS4A family members but not MS4A8) to confirm the specificity profile of the designed antibodies .

How can MS4A8 antibodies be implemented in studies of neurodegenerative diseases?

MS4A8 antibodies offer valuable tools for investigating neurodegenerative pathologies, particularly given the emerging link between the MS4A gene cluster and Alzheimer's disease:

• Clinical sample analysis: MS4A8 antibodies can be used to assess protein expression in post-mortem brain tissue from neurodegenerative disease patients compared to age-matched controls using immunohistochemistry or Western blotting.

• Genetic correlation studies: For individuals with known MS4A gene locus variants (e.g., rs1582763, rs6591561), MS4A8 antibodies can quantify protein expression to establish genotype-phenotype correlations. Research has shown that these variants are associated with altered CSF sTREM2 levels and modified Alzheimer's disease risk .

• Cell-type specific analysis: Using MS4A8 antibodies in combination with cell-type markers (e.g., microglia, macrophages) can reveal cell-specific expression patterns and potential alterations in disease states.

• Temporal expression profiling: MS4A8 antibodies can track expression changes throughout disease progression in animal models of neurodegeneration, potentially revealing critical timepoints for therapeutic intervention.

• Functional assays: Antibodies can be used to block MS4A8 function in cellular assays to assess its role in neuroinflammatory responses, similar to approaches that have demonstrated MS4A4A's role in modulating sTREM2 production .

When designing such studies, researchers should account for potential regional variations in MS4A8 expression within the brain and consider the genetic background of the samples, particularly regarding known MS4A locus variants that affect expression and function.

What are common technical challenges when using MS4A8 antibodies, and how can they be addressed?

Researchers frequently encounter several challenges when working with MS4A8 antibodies that require specific troubleshooting approaches:

Challenge 1: Weak or Absent Signal

• Cause: Insufficient protein expression, epitope masking, or improper antibody concentration

• Solution: Optimize antibody dilution (try 1:100-1:500 for WB) , enhance antigen retrieval methods, confirm MS4A8 expression in sample, and use fresh samples or positive controls like U-87MG cells

Challenge 2: High Background Signal

• Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

• Solution: Increase blocking time/concentration (5% BSA recommended), optimize antibody dilution, increase wash frequency/duration, and consider using purified antibody fractions

Challenge 3: Non-specific Bands in Western Blot

• Cause: Cross-reactivity with related MS4A family members, sample degradation, or non-specific binding

• Solution: Use antibodies targeting unique epitopes (e.g., N-terminal regions) , include protease inhibitors during sample preparation, optimize blocking, and consider more stringent washing conditions

Challenge 4: Inconsistent Immunostaining Patterns

• Cause: Variable fixation conditions, processing artifacts, or antibody batch variation

• Solution: Standardize fixation protocols, perform antigen retrieval optimization, and validate each antibody batch with positive and negative controls

Challenge 5: Epitope Accessibility Issues

• Cause: MS4A8's multi-pass membrane topology may obscure epitopes

• Solution: For membrane proteins like MS4A8, ensure adequate membrane permeabilization using appropriate detergents (0.1-0.5% Triton X-100 or 0.1% saponin), and select antibodies targeting accessible regions (N-terminal epitopes have shown success)

Documenting troubleshooting steps and results systematically will help establish optimal protocols for consistent MS4A8 detection across experiments.

How can cross-reactivity issues with other MS4A family members be addressed?

Cross-reactivity with other MS4A family members is a significant concern given their sequence homology and shared structural features:

• Epitope selection: Choose antibodies targeting regions with minimal homology to other MS4A family members. The N-terminal regions (e.g., AA 44-70) generally offer greater differentiation, while transmembrane domains tend to be more conserved .

• Absorption controls: Pre-incubate the antibody with recombinant proteins of related MS4A family members to identify potential cross-reactivity. If signal is reduced after pre-incubation with non-target MS4A proteins, cross-reactivity is likely occurring.

• Knockout/knockdown validation: MS4A8-specific siRNA/shRNA can create targeted knockdown samples for validation. An effective MS4A8 antibody should show reduced signal in MS4A8 knockdown samples but unchanged signal in samples with knockdown of other MS4A family members .

• Orthogonal detection methods: Combine antibody-based detection with mRNA quantification (qPCR with gene-specific primers) to confirm specificity. MS4A8-specific mRNA levels should correlate with protein detection patterns if the antibody is specific.

• Multiple antibody approach: Use two antibodies targeting different MS4A8 epitopes; consistent detection patterns increase confidence in specificity.

• Western blot molecular weight verification: MS4A8 has a molecular weight of approximately 26 kDa . Careful analysis of band size can help distinguish it from other MS4A family members that may have different molecular weights.

When publishing results, always specify the validation steps performed to confirm antibody specificity, as this significantly enhances data interpretation and reproducibility.

What considerations are important when using MS4A8 antibodies in multiplexed immunoassays?

Multiplexed immunoassays that simultaneously detect MS4A8 alongside other proteins require specific technical considerations:

• Antibody species and isotype selection: When designing multiplexed panels, select MS4A8 antibodies from different host species than other target antibodies (rabbit polyclonal vs. mouse monoclonal) to enable clear distinction with secondary antibodies. Available MS4A8 antibodies include rabbit polyclonal and mouse monoclonal (clone 1B4) options.

• Fluorophore selection and spectral overlap: When using fluorescently conjugated MS4A8 antibodies (e.g., FITC-conjugated variants) , carefully assess spectral overlap with other fluorophores in your panel and implement appropriate compensation controls.

• Sequential staining approach: For challenging multiplexed applications, consider sequential staining protocols where the MS4A8 antibody incubation and detection are performed separately from other antibodies, with an intermediate blocking step.

• Cross-reactivity testing: Before full implementation, test each antibody individually and in combination to identify any unexpected cross-reactivity or interference between detection systems.

• Signal amplification balancing: If MS4A8 expression is substantially different from other targets, balance detection sensitivity through dilution adjustments or selective amplification of weaker signals.

• Quantification standardization: For quantitative multiplexed assays, include calibration standards for each target, including MS4A8, to enable accurate cross-comparison of expression levels.

• Data analysis approaches: Employ multiparameter analysis tools that can address potential bleed-through or interference between channels, particularly important when working with closely related family members that might be included in the same panel.

By carefully addressing these considerations, researchers can successfully incorporate MS4A8 detection into multiplexed immunoassay panels for comprehensive protein expression analysis.

How should researchers quantify and normalize MS4A8 expression data across different experimental platforms?

Accurate quantification and normalization of MS4A8 expression requires platform-specific approaches:

Western Blot Quantification:

• Use densitometry software to quantify band intensity relative to loading controls

• Normalize MS4A8 signal to housekeeping proteins (β-actin, GAPDH) for whole-cell lysates

• For membrane fraction analysis, normalize to membrane-specific controls (Na+/K+ ATPase, calnexin)

• Include a standard curve using recombinant MS4A8 when absolute quantification is required

Immunohistochemistry Quantification:

• Employ digital image analysis software to quantify staining intensity

• Use H-scoring (combining intensity and percentage of positive cells)

• For tissue microarrays, normalize to control tissues included on the same slide

• The Human Protein Atlas provides standardized scoring methods for MS4A8 that can be adopted for consistency

Flow Cytometry Quantification:

• Report data as median fluorescence intensity (MFI) or percentage of positive cells

• Use isotype controls to set positive/negative thresholds

• For absolute quantification, include calibration beads with known antibody binding capacity

ELISA Quantification:

• Generate standard curves using recombinant MS4A8 protein

• For cell/tissue lysates, normalize to total protein concentration

• Use recommended dilutions (e.g., 1:1000 for MS4A8 ELISA applications)

Cross-Platform Normalization:

• Include common samples across platforms as bridging controls

• Convert measurements to fold-change relative to a standard reference sample

• When comparing across studies, focus on relative changes rather than absolute values unless standardized protocols were used

When publishing, clearly document quantification methods, software used, normalization strategies, and statistical approaches to enable reproducibility.

What information can expression patterns of MS4A8 reveal about biological function and disease association?

MS4A8 expression patterns provide valuable insights into its biological functions and disease associations:

• Tissue Distribution Pattern: MS4A8 is primarily expressed in hematopoietic tissues and cell lines . Deviations from this pattern may indicate pathological conditions or adaptive responses.

• Correlation with MS4A Genetic Variants: Expression levels of MS4A8 may vary with MS4A locus genetic variants. For example, the rs1582763 variant is associated with altered expression in the MS4A gene cluster and modified Alzheimer's disease risk .

• Co-expression Patterns: MS4A8 co-expression with other immune regulators, particularly TREM2, may indicate functional relationships in immune regulation and neuroinflammation. Research has shown that MS4A family members can modulate soluble TREM2 levels, which are relevant to Alzheimer's disease pathogenesis .

• Cellular Localization: As a multi-pass membrane protein, MS4A8 is expected to localize to the plasma membrane, potentially in specialized microdomains. Altered subcellular localization may indicate dysfunction or regulation.

• Immune Cell Type Distribution: The pattern of expression across immune cell subsets can reveal functional specialization. MS4A family members are expressed in microglia, suggesting roles in neuroinflammatory processes .

• Disease-Associated Changes: Altered MS4A8 expression in specific diseases, particularly neurodegenerative conditions, may indicate pathological involvement. The MS4A gene cluster has been implicated in Alzheimer's disease through genome-wide association studies .

• Response to Stimuli: Changes in MS4A8 expression following immune stimulation, inflammatory challenges, or disease progression may reveal regulatory mechanisms and functional roles.

By systematically analyzing these expression patterns in normal and pathological contexts, researchers can generate hypotheses about MS4A8's functional roles and potential as a biomarker or therapeutic target, particularly in neuroinflammatory and neurodegenerative diseases.

How do MS4A8 protein variants and post-translational modifications affect antibody detection?

MS4A8 protein variants and post-translational modifications can significantly impact antibody detection and require careful consideration when interpreting experimental results:

Genetic Variants:

• Missense Variants: Variants like rs6591561 (MS4A4A p.M159V) in the related MS4A4A protein affect function and detection . Similar variants in MS4A8 could alter epitope structure and antibody binding.

• Splice Variants: The Human Protein Atlas acknowledges multiple splice variants of MS4A8 , which may lack specific epitopes targeted by antibodies. Antibodies targeting different regions should be selected based on which variant is of interest.

• Epitope-Specific Impacts: Genetic variations within antibody epitopes (e.g., within the N-terminal AA 44-70 region) would have greater impacts on detection than variants in other regions .

Post-Translational Modifications (PTMs):

• Glycosylation: As a membrane protein, MS4A8 may be glycosylated, which can mask epitopes or alter apparent molecular weight in Western blots. Deglycosylation treatments before analysis may be necessary for consistent detection.

• Phosphorylation: Signal transduction proteins often undergo regulatory phosphorylation. Phosphorylation within antibody epitopes may prevent antibody binding, particularly for antibodies raised against non-phosphorylated peptides.

• Proteolytic Processing: Cleavage events may separate epitopes from the remainder of the protein. Antibodies targeting different regions (N-terminal vs. internal) may give discrepant results if proteolytic processing occurs.

Methodological Approaches:

• Multiple Antibody Strategy: Use antibodies targeting different MS4A8 epitopes to comprehensively detect all variants and modified forms.

• Sample Treatment Controls: Include controls with and without treatments that remove PTMs (e.g., phosphatase, deglycosylation enzymes) to assess their impact on detection.

• Mass Spectrometry Validation: For critical studies, complement antibody-based detection with mass spectrometry to identify specific variants and PTMs present in samples.

• Recombinant Control Proteins: Include controls using recombinant MS4A8 with known modifications to calibrate detection sensitivity.

When publishing results, document the specific antibody epitope used and acknowledge that detection may be influenced by variants or modifications that affect that particular region of MS4A8.