MS4A2 Antibody

What is the molecular structure and function of MS4A2?

MS4A2 (membrane-spanning 4-domains, subfamily A, member 2) is a component of the high-affinity IgE receptor, also known as the beta-chain of FcεRIβ. The protein contains four transmembrane-spanning domains and is crucial for cell differentiation, signaling, and cell cycle control. Co-immunoprecipitation experiments have demonstrated that MS4A2 associates with FcɛRIα and FcRγ to form a tetrameric receptor complex (αβγ2) that is expressed on mast cells and basophils at high density . The calculated molecular weight is 27 kDa, with observed molecular weights in assays ranging from 25-27 kDa . This protein plays a critical role in the allergic response by enhancing receptor maturation and signal transduction capacity.

How does MS4A2 function in the allergic cascade?

MS4A2 functions as the beta subunit of the high-affinity IgE receptor, which is central to allergic responses. The complete IgE receptor is a tetramer composed of an alpha, beta, and two disulfide-linked gamma chains found on the surface of mast cells and basophils . In allergic reactions, allergen binding to receptor-bound IgE triggers cell activation and the release of mediators that drive allergic manifestations . The beta-chain specifically enhances receptor maturation and signal transduction capacity, leading to the release of proinflammatory mediators and cytokines that can exacerbate asthma symptoms . Research has shown that polymorphisms in MS4A2, particularly the FcepsilonR1beta-109T>C variant, may increase MS4A2 expression in mast cells, leading to enhanced release of proinflammatory mediators in asthmatic airways .

What is the interspecies homology of MS4A2?

The conservation of MS4A2 across species is an important consideration for experimental design and antibody selection. Based on available data, the mouse MS4A2 shares approximately 57% sequence homology with human MS4A2, while rat MS4A2 shares approximately 55% homology with the human protein . This moderate level of homology has implications for cross-reactivity of antibodies across species and the translational relevance of animal model findings to human conditions.

What are the optimal conditions for Western Blot detection of MS4A2?

For Western Blot applications, the recommended dilution range for MS4A2 antibodies is typically 1:1000-1:4000 . The expected molecular weight band should appear between 25-27 kDa. For optimal results, researchers should consider tissue-specific expression patterns, with positive Western Blot detection confirmed in mouse lung tissue . When optimizing Western Blot protocols for MS4A2:

• Sample preparation should include appropriate lysis buffers that effectively solubilize membrane proteins while preserving epitope integrity

• Include appropriate controls (positive tissue samples such as lung tissue)

• Consider running gradient gels (10-15%) to optimize resolution in the 25-27 kDa range

• Blocking solutions may need optimization depending on the specific antibody used (typically 5% BSA or non-fat milk)

• Titration of the antibody within the recommended range is advised for each experimental system to obtain optimal signal-to-noise ratio

How should ELISA protocols be optimized for MS4A2 detection?

For quantitative detection of MS4A2 protein levels, sandwich ELISA provides sensitivity ranging from 3 ng/ml to 100 ng/ml . When setting up an ELISA protocol:

• Use a matched antibody pair system with:

  • Capture antibody: rabbit affinity-purified polyclonal anti-MS4A2

  • Detection antibody: mouse monoclonal anti-MS4A2 (IgG2a Kappa)

• Generate a standard curve using recombinant MS4A2 protein as an analyte

• Optimize blocking reagents to minimize background signal

• Consider sample dilution series to ensure readings fall within the linear range of the standard curve

• Calculate protein concentration using a four-parameter logistic curve fit

The reagents provided in commercial antibody pair sets are typically sufficient for at least 1-2 x 96 well plates using recommended protocols .

What considerations are important for immunohistochemistry and immunofluorescence applications?

MS4A2 antibodies have demonstrated utility in immunohistochemistry (IHC) and immunofluorescence (IF) applications . For these applications, consider:

• Fixation method: Paraformaldehyde (4%) is often suitable for maintaining MS4A2 epitope accessibility

• Antigen retrieval may be necessary depending on the fixation method and specific antibody

• Expression pattern should be evaluated against known biology - MS4A2 is predominantly expressed on mast cells and basophils

• Proper controls should include:

  • Negative controls (secondary antibody only)

  • Positive tissue controls (tissues known to express MS4A2)

  • Blocking peptide controls to confirm specificity

For co-localization studies, MS4A2 antibodies can be paired with markers for mast cells and basophils to confirm cell type-specific expression patterns.

How can MS4A2 antibodies be used to study allergy and asthma mechanisms?

MS4A2 antibodies can serve as valuable tools for investigating the molecular mechanisms underlying allergic disorders and asthma. Research has demonstrated that the FcepsilonR1beta-109T>C polymorphism influences MS4A2 expression and is associated with aspirin-intolerant asthma . Experimental approaches include:

• Comparing MS4A2 protein levels in clinical samples from patients with different asthma phenotypes

• Assessing the correlation between MS4A2 expression and specific IgE levels to allergens, particularly Staphylococcal enterotoxin B (SEB)

• Investigating the impact of genetic polymorphisms on MS4A2 protein expression through luciferase reporter assays

• Analyzing the formation and function of the high-affinity IgE receptor complex through co-immunoprecipitation studies

• Evaluating the effects of therapeutic interventions on MS4A2 expression and signaling in mast cells

Researchers found that the FcepsilonR1beta-109T allele was associated with higher promoter activity of MS4A2 in both RBL-2H3 and A549 cell lines, providing a molecular mechanism for the enhanced allergic response observed clinically .

What is the role of MS4A2 in cancer biology, particularly lung adenocarcinoma?

Emerging research has identified MS4A2 as being strongly associated with the prognosis of lung adenocarcinoma and lung cancer brain metastases . Investigators studying this connection should consider:

• Comparative analysis of MS4A2 expression levels between cancerous and non-cancerous lung tissues

• Correlation of MS4A2 expression with clinical outcomes and survival data

• Investigation of potential mechanisms by which MS4A2 influences tumor progression

• Assessment of whether tumor-associated mast cells in the lung microenvironment express altered levels of MS4A2

• Evaluation of MS4A2 as a potential biomarker for predicting metastatic potential, particularly to the brain

The association of MS4A2 with cancer prognosis suggests that antibodies targeting this protein may have utility in both basic cancer research and potential translational applications.

How can contradictory results in MS4A2 research be reconciled?

When encountering contradictory results in MS4A2 research, consider the following methodological factors:

• Antibody specificity - confirm the exact epitope recognized by different antibodies

• Cell/tissue context - MS4A2 may function differently depending on the cellular environment

• Genetic background - polymorphisms in the MS4A2 gene may affect protein function and expression

• Experimental conditions - differences in experimental design, including sample preparation and detection methods

• Species differences - the moderate homology between human and rodent MS4A2 (55-57%) may lead to species-specific findings

To reconcile conflicting results, design experiments with appropriate controls and directly compare methodologies from previous studies to identify sources of variation.

How should problems with antibody cross-reactivity be addressed?

Cross-reactivity concerns can arise due to the structural similarity of MS4A family members or other proteins. To address this:

• Validate antibody specificity through:

  • Western blot analysis of recombinant MS4A2 protein

  • Knockdown/knockout validation using siRNA or CRISPR

  • Competitive blocking with immunizing peptide

  • Parallel testing with multiple antibodies against different MS4A2 epitopes

• Consider potential cross-reactivity with other MS4A family members (MS4A1, MS4A3, etc.)

• Be aware that different antibody dilutions may be required to minimize cross-reactivity while maintaining specific signal

• Consult antibody validation data for each specific application (WB, IHC, IF, ELISA)

What are the best approaches for optimizing signal-to-noise ratio in MS4A2 detection?

To improve signal-to-noise ratio when working with MS4A2 antibodies:

• For Western blots:

  • Optimize blocking conditions (BSA vs. milk, concentration, blocking time)

  • Titrate primary antibody concentration within recommended range (1:1000-1:4000)

  • Consider longer but more dilute primary antibody incubation (overnight at 4°C)

  • Use high-sensitivity detection systems for low abundance samples

  • Include appropriate washing steps (at least 3×10 minutes between antibodies)

• For immunostaining:

  • Test multiple antigen retrieval methods

  • Use autofluorescence reduction techniques for IF applications

  • Consider signal amplification methods for low-abundance detection

  • Optimize antibody concentration through titration experiments

  • Use tissue-specific positive controls to benchmark signal intensity

• For ELISA:

  • Optimize coating buffer composition and concentration

  • Test different blocking reagents (BSA, casein, commercial blockers)

  • Carefully validate sample dilution to ensure measurements within the linear range

  • Consider using streptavidin-HRP systems for enhanced sensitivity

How should MS4A2 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of MS4A2 antibodies is crucial for maintaining their activity and specificity:

• Store antibodies at -20°C for long-term storage, where they are typically stable for one year after shipment

• For antibodies in storage buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3, aliquoting is unnecessary for -20°C storage

• Avoid repeated freeze-thaw cycles by creating appropriate working aliquots

• Return reagents to -20°C storage immediately after use

• For short-term storage (1-2 weeks), antibodies can typically be kept at 4°C

• Be aware that some antibody preparations may contain BSA (0.1%) as a stabilizer

• Avoid exposure to light for fluorophore-conjugated antibodies

• Follow manufacturer's recommendations for each specific antibody formulation

What controls should be included in MS4A2 antibody-based experiments?

A robust experimental design for MS4A2 research should include the following controls:

• Positive tissue controls:

  • Mouse lung tissue has been validated for Western blot applications

  • Human/mouse mast cell or basophil preparations

  • Cell lines known to express MS4A2 (e.g., RBL-2H3)

• Negative controls:

  • Tissues or cells known not to express MS4A2

  • Secondary antibody-only controls

  • Isotype controls for monoclonal antibodies

• Validation controls:

  • MS4A2 knockdown/knockout samples

  • Blocking peptide competition

  • Multiple antibodies targeting different epitopes

• Loading/processing controls:

  • Housekeeping proteins for Western blots

  • Cellular markers for immunostaining

  • Standard curves for quantitative applications

How should researchers design experiments to study MS4A2 polymorphisms?

When investigating MS4A2 polymorphisms, particularly those with functional consequences like FcepsilonR1beta-109T>C:

• Design genotyping assays with appropriate primers and probes

• Include population-stratified sampling to account for ethnic differences in polymorphism frequency

• Consider functional validation through:

  • Luciferase reporter assays to assess promoter activity differences

  • Expression analysis in patient-derived samples

  • Correlation with clinical phenotypes (e.g., aspirin sensitivity in asthma)

• Include appropriate sample sizes based on power calculations considering polymorphism frequency

• Analyze gene-environment interactions that may influence the phenotypic expression of polymorphisms

• Consider the impact of polymorphisms on protein-protein interactions within the IgE receptor complex

What approaches can be used to study MS4A2 in the context of receptor complex formation?

To investigate MS4A2's role in forming the high-affinity IgE receptor complex:

• Co-immunoprecipitation studies to confirm interactions with FcɛRIα and FcRγ subunits

• Proximity ligation assays to visualize protein-protein interactions in situ

• FRET/BRET approaches to measure dynamic interactions between receptor components

• Site-directed mutagenesis to identify critical domains for complex assembly

• Live-cell imaging to track receptor complex formation and trafficking

• Cross-linking studies to stabilize transient interactions before analysis

• Expression of fluorescently-tagged components to track co-localization and assembly

Understanding the tetrameric receptor complex (αβγ2) formation is essential for comprehending MS4A2's role in allergic responses and mast cell activation.