MEX3B Antibody

What is MEX3B and why is it important in research?

MEX3B (mex-3 RNA binding family member B) is a multifunctional RNA-binding protein that plays significant roles in post-transcriptional regulation and immune response pathways. It is approximately 58.8 kilodaltons in mass and contains two K homology (KH) domains that bind RNA, plus a RING finger domain with potential E3 ubiquitin ligase activity . MEX3B is particularly important in research because it functions as a coreceptor for Toll-like receptor 3 (TLR3) in innate antiviral responses, binding to double-stranded RNA (dsRNA) and enhancing TLR3-mediated signaling . This critical immune function makes MEX3B a valuable target for immunology research, especially studies examining antiviral immunity and inflammatory response mechanisms.

What are the alternative names for MEX3B protein?

When conducting literature searches or selecting antibodies for MEX3B, researchers should be aware of its alternative nomenclature to ensure comprehensive coverage of available information. MEX3B may be referenced in literature and product catalogs under several alternative designations including:

• MEX-3B

• RKHD3 (Ring finger and KH domain containing 3)

• RNF195 (RING finger protein 195)

• RNA-binding protein MEX3B

Including these alternative names in search strategies will help ensure that relevant research materials and literature are not overlooked.

How do I select the appropriate MEX3B antibody for my specific research application?

Selecting the optimal MEX3B antibody requires careful consideration of several experimental parameters:

• Application compatibility: Determine whether the antibody has been validated for your specific application (WB, ELISA, IF, IHC, IP). Not all antibodies perform consistently across different techniques .

• Species reactivity: Verify reactivity with your experimental species. While many MEX3B antibodies react with human samples, if working with mouse or rat models, confirm cross-reactivity with these species .

• Epitope specificity: Consider whether you need an antibody that targets a specific domain of MEX3B (e.g., N-terminal, internal region, or KH domains).

• Clonality: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies provide broader recognition but potentially more background.

• Conjugation requirements: Determine if you need a conjugated antibody (FITC, HRP, Alexa Fluor) for direct detection or an unconjugated primary antibody .

The table below summarizes key considerations for common research applications:

What are the recommended protocols for using MEX3B antibodies in Western blot experiments?

When using MEX3B antibodies for Western blot analysis, researchers should follow these methodological recommendations:

• Sample preparation: Total cell lysates work well, but nuclear and cytoplasmic fractionation may provide more specific localization information. Use RIPA buffer with protease inhibitors for extraction.

• Protein loading: Load 20-50 μg of total protein per lane. MEX3B is expressed at moderate levels in most cells.

• Gel separation: Use 10% SDS-PAGE gels for optimal separation around the 58.8 kDa range of MEX3B .

• Transfer conditions: Semi-dry or wet transfer at 100V for 1-2 hours works well for MEX3B.

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute antibody according to manufacturer's recommendation (typically 1:500-1:2000) in 5% BSA/TBST and incubate overnight at 4°C.

• Expected band size: The main MEX3B band should appear at approximately 58.8 kDa , though post-translational modifications may result in additional bands.

• Controls: Include a positive control (cell line known to express MEX3B) and consider using MEX3B knockout or knockdown samples as negative controls to validate specificity.

How should I optimize immunofluorescence staining with MEX3B antibodies?

Optimizing immunofluorescence staining with MEX3B antibodies requires attention to several methodological details:

• Fixation method: Compare 4% paraformaldehyde (10-15 minutes) with methanol fixation (-20°C, 10 minutes) to determine which best preserves MEX3B epitopes without compromising cellular structure.

• Permeabilization: Use 0.1-0.3% Triton X-100 in PBS for 5-10 minutes. For nuclear MEX3B detection, ensure adequate nuclear permeabilization.

• Blocking: Block with 5% normal serum (matched to secondary antibody host) with 0.1% Triton X-100 in PBS for 1 hour at room temperature.

• Antibody dilution: Start with the manufacturer's recommended dilution, typically 1:100-1:500 for immunofluorescence applications .

• Subcellular localization: MEX3B typically shows both cytoplasmic and nuclear localization, with enrichment in endosomal compartments where it interacts with TLR3 .

• Co-localization studies: Consider dual staining with endosomal markers (EEA1, LAMP1) to verify MEX3B's association with endosomes, or with TLR3 to demonstrate their interaction .

• Signal amplification: If signal is weak, try tyramide signal amplification or use a conjugated secondary antibody with bright fluorophores like Alexa Fluor 488 or 594.

What controls should be included when validating a new MEX3B antibody?

Proper validation of a new MEX3B antibody requires a comprehensive set of controls:

• Positive expression controls:

  • Cell lines with known MEX3B expression (based on literature or database information)

  • Tissues with documented MEX3B expression

  • MEX3B overexpression systems (transfected cell lines)

• Negative controls:

  • MEX3B knockout or knockdown (siRNA/shRNA) samples

  • Cell lines with minimal MEX3B expression

  • Primary antibody omission control

  • Isotype control antibody

• Specificity controls:

  • Peptide competition assay using the immunizing peptide

  • Testing multiple antibodies targeting different epitopes of MEX3B

  • Western blot to confirm a single band at the expected molecular weight (58.8 kDa)

• Cross-reactivity assessment:

  • Testing in multiple species if cross-reactivity is claimed

  • Checking for cross-reactivity with other MEX3 family members (MEX3A, MEX3C, MEX3D)

A systematic validation approach not only confirms antibody specificity but also establishes optimal working conditions for each application.

How can MEX3B antibodies be used to study TLR3-mediated antiviral responses?

MEX3B antibodies are valuable tools for investigating TLR3-mediated antiviral responses due to MEX3B's role as a TLR3 coreceptor . Here are methodological approaches:

• Co-immunoprecipitation studies: Use MEX3B antibodies to pull down protein complexes from cells stimulated with poly(I:C) or viral dsRNA, followed by blotting for TLR3 and other signaling components to map interaction networks .

• Proximity ligation assay (PLA): Employ MEX3B antibodies alongside TLR3 antibodies in PLA to visualize and quantify endogenous protein-protein interactions in situ, particularly in endosomal compartments.

• ChIP-seq analysis: Utilize MEX3B antibodies for chromatin immunoprecipitation followed by sequencing to identify genome-wide binding patterns following viral infection or poly(I:C) stimulation.

• Subcellular fractionation with immunoblotting: Combine organelle isolation techniques with MEX3B antibodies to track dynamic localization changes during viral infection.

• FACS-based analysis: Use fluorescently-conjugated MEX3B antibodies for flow cytometry to quantify expression changes in different immune cell populations during antiviral responses.

Research has demonstrated that MEX3B is required for dsRNA-induced, TLR3-mediated activation of antiviral genes. Knockout studies showed that Mex3b-/- mice exhibited reduced production of IFN-β in response to poly(I:C) and were more resistant to poly(I:C)-induced death, highlighting the critical role of MEX3B in this pathway .

What is known about the interaction between MEX3B and TLR3, and how can this be investigated using antibodies?

The interaction between MEX3B and TLR3 represents a critical mechanism in innate immunity. MEX3B has been shown to:

• Associate with TLR3 specifically in endosomal compartments

• Bind to dsRNA directly and enhance TLR3's dsRNA-binding activity

• Promote the proteolytic processing of TLR3, which is essential for receptor activation

To investigate these interactions using antibodies, researchers can employ:

Methodological approach 1: Co-immunoprecipitation with domain mapping

• Immunoprecipitate MEX3B using validated antibodies and blot for TLR3

• Use domain-specific antibodies or recombinant protein fragments to determine which domains of MEX3B (KH domains vs. RING finger domain) are essential for TLR3 interaction

• Perform reciprocal IP with TLR3 antibodies to confirm the interaction

Methodological approach 2: Functional studies with neutralizing antibodies

• Utilize antibodies targeting specific epitopes of MEX3B to disrupt its interaction with TLR3

• Measure downstream signaling (NF-κB activation, IFN-β production) to assess functional consequences

• Compare effects with targeted disruption using domain-specific mutants lacking RNA-binding activity

Methodological approach 3: Super-resolution microscopy

• Use dual immunofluorescence with MEX3B and TLR3 antibodies

• Apply structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to visualize co-localization at endosomal membranes with nanometer resolution

• Track dynamic interactions following poly(I:C) stimulation using live-cell imaging

Research findings have demonstrated that mutants of MEX3B lacking RNA-binding activity inhibited TLR3-mediated IFN-β induction, suggesting that the RNA-binding property of MEX3B is essential for its function as a TLR3 coreceptor .

How does MEX3B expression change in different pathological conditions, and what antibody-based methods can be used to study these changes?

MEX3B expression patterns change in various pathological states, particularly during viral infections and in certain cancers. Antibody-based methods provide powerful tools to track these changes:

• Immunohistochemistry of tissue microarrays: Use validated MEX3B antibodies on tissue microarrays containing samples from normal and diseased tissues to generate quantitative expression profiles across pathological conditions.

• Multiplex immunofluorescence: Combine MEX3B antibodies with markers of cell type, activation status, and viral infection to characterize expression patterns in complex tissue environments.

• Quantitative flow cytometry: Use fluorescently-labeled MEX3B antibodies to measure expression levels in different immune cell subsets during infection or inflammatory conditions.

• Tissue-based proteomics: Apply MEX3B antibodies in techniques like imaging mass cytometry or multiplexed ion beam imaging to obtain spatially resolved proteomic data from tissue sections.

• Single-cell western blot: Employ MEX3B antibodies in microfluidic single-cell western blot systems to study cell-to-cell variability in expression during pathological processes.

To quantify changes accurately, researchers should:

• Use image analysis software with standardized quantification parameters

• Include multiple antibody clones targeting different epitopes

• Normalize MEX3B expression to appropriate housekeeping proteins

• Employ statistical methods appropriate for comparing expression across multiple samples and conditions

Why might I see multiple bands when using MEX3B antibodies in Western blot, and how should I interpret them?

Multiple bands in MEX3B Western blots can occur for several biological and technical reasons. Here's how to interpret and address this common issue:

Potential biological explanations:

• Alternative splicing: MEX3B may exist in multiple splice variants, generating proteins of different molecular weights.

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can shift the apparent molecular weight of MEX3B. The RING finger domain suggests potential auto-ubiquitination activity.

• Proteolytic processing: As MEX3B promotes the proteolytic processing of TLR3 , it might itself undergo processing during activation.

• Family member cross-reactivity: Antibodies may cross-react with other MEX3 family proteins (MEX3A, MEX3C, MEX3D) that share sequence homology.

Methodological approaches to resolve and interpret multiple bands:

• Confirm specificity through knockout/knockdown: Compare Western blots of wild-type samples with MEX3B knockout or knockdown samples to identify which bands disappear.

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to determine which bands represent specific binding.

• Subcellular fractionation: Separate nuclear, cytoplasmic, and membrane fractions to determine if different forms of MEX3B localize to different cellular compartments.

• Phosphatase treatment: Treat samples with lambda phosphatase to determine if higher molecular weight bands are due to phosphorylation.

• Use multiple antibodies: Test antibodies targeting different epitopes of MEX3B to build a consensus pattern of specific bands.

What are the common pitfalls when using MEX3B antibodies, and how can they be avoided?

Researchers working with MEX3B antibodies should be aware of these common pitfalls and employ the corresponding mitigation strategies:

Additionally, when comparing MEX3B expression between different experimental conditions, always include relevant loading controls and consider quantifying protein levels across multiple experiments to account for variability.

How can I determine if my MEX3B antibody is detecting the functionally active form of the protein?

Determining whether an MEX3B antibody detects the functionally active form requires experimental approaches that link antibody recognition to protein function:

• Phospho-specific antibody analysis: Investigate whether MEX3B undergoes activity-dependent phosphorylation by comparing standard and phospho-specific MEX3B antibodies following stimulation with poly(I:C) or viral infection.

• Correlation with downstream signaling: Stimulate cells with poly(I:C) and perform time-course analysis correlating MEX3B detection patterns with activation of downstream signaling molecules (phospho-IRF3, IFN-β production).

• Association with functional complexes: Use size-exclusion chromatography or sucrose gradient fractionation followed by immunoblotting to determine if the antibody recognizes MEX3B in high-molecular-weight complexes that represent active signaling platforms.

• Subcellular localization during activation: Active MEX3B should co-localize with TLR3 in endosomal compartments during dsRNA stimulation . Use immunofluorescence to confirm that the antibody detects MEX3B in these functionally relevant locations.

• RNA-binding activity assessment: Combine RNA immunoprecipitation with MEX3B antibodies followed by RT-PCR or sequencing to verify that the detected protein possesses RNA-binding activity, which is essential for its function .

• Co-detection with functional partners: Perform proximity ligation assays to verify that the antibody detects MEX3B when it's in close proximity to known functional partners like TLR3.

Research has shown that mutants of MEX3B lacking RNA-binding activity inhibit TLR3-mediated IFN-β induction , suggesting that RNA binding is critical for MEX3B function. Antibodies that can distinguish between RNA-bound and unbound forms would be particularly valuable for functional studies.

How can MEX3B antibodies be used to explore novel therapeutic targets in antiviral immunity?

MEX3B antibodies offer powerful tools for investigating potential therapeutic targets in antiviral immunity, particularly given MEX3B's role as a TLR3 coreceptor . Here are methodological approaches for such explorations:

• Target validation in primary human cells: Use MEX3B antibodies to assess expression levels across different immune cell populations isolated from healthy donors and virus-infected patients to identify cell types where therapeutic targeting would be most relevant.

• Small molecule compound screening: Develop high-throughput screening assays using MEX3B antibodies to detect conformational changes or altered localization following compound treatment, identifying molecules that modulate MEX3B function.

• Structure-function relationships: Combine epitope-specific MEX3B antibodies with mutagenesis studies to map critical functional domains that could be targeted by therapeutics without disrupting essential cellular functions.

• Patient stratification biomarkers: Evaluate MEX3B expression patterns in patient samples to determine if expression levels or post-translational modifications correlate with disease severity or treatment response.

• Nanobody development: Engineer single-domain antibodies (nanobodies) against specific MEX3B epitopes that can be used as both research tools and potential therapeutic agents to modulate MEX3B function in vivo.

Research findings indicate that Mex3b-/- mice were more resistant to poly(I:C)-induced death , suggesting that targeted modulation of MEX3B activity might provide therapeutic benefits in conditions characterized by excessive TLR3 activation and inflammatory response.

What are the emerging techniques for studying MEX3B interactions that require specialized antibodies?

Several cutting-edge techniques are expanding our understanding of MEX3B biology, each requiring specifically optimized antibodies:

• Proximity-dependent biotinylation (BioID/TurboID):

  • Requires antibodies that recognize MEX3B fusion proteins without cross-reactivity

  • Enables identification of the proximal protein interactome in living cells

  • Can reveal transient interactions missed by traditional co-IP approaches

• CRISPR epitope tagging combined with antibody detection:

  • Allows visualization and quantification of endogenous MEX3B dynamics

  • Requires highly specific antibodies against small epitope tags

  • Enables tracking of MEX3B without overexpression artifacts

• Super-resolution microscopy:

  • Demands antibodies with minimal background and high signal-to-noise ratio

  • Permits visualization of MEX3B localization with nanometer precision

  • Can resolve co-localization with TLR3 at the single-molecule level

• Mass spectrometry-based interactomics:

  • Requires antibodies suitable for immunoprecipitation under native conditions

  • Allows identification of MEX3B interaction partners and post-translational modifications

  • Can detect dynamic changes in the MEX3B interactome following stimulation

• Single-cell proteomics with antibody-based detection:

  • Needs antibodies validated for flow cytometry or mass cytometry (CyTOF)

  • Enables correlation of MEX3B expression with cellular phenotypes at single-cell resolution

  • Can identify rare cell populations with unique MEX3B expression patterns

For these advanced applications, researchers should consider using recombinant antibodies, which offer improved batch-to-batch consistency and can be engineered for optimal performance in specific applications.

What are the best practices for documenting MEX3B antibody usage in publications?

To enhance reproducibility and enable proper interpretation of MEX3B research findings, authors should adhere to these documentation standards when publishing studies using MEX3B antibodies:

• Comprehensive antibody identification:

  • Manufacturer and catalog number

  • Clone designation for monoclonal antibodies

  • Lot number (particularly important for polyclonal antibodies)

  • RRID (Research Resource Identifier) when available

• Validation evidence:

  • Description of controls used (positive, negative, specificity)

  • Reference to validation experiments or citation of prior validation

  • Western blot images showing specificity when introducing a new antibody

  • For critical findings, validation using genetic approaches (knockout/knockdown)

• Detailed methods:

  • Complete protocol including dilutions, incubation times and temperatures

  • Buffer compositions and blocking reagents

  • Antigen retrieval methods for IHC/IF

  • Secondary antibody details and detection systems

• Quantification approaches:

  • Methods used to quantify signals (densitometry, fluorescence intensity)

  • Software used for image analysis and specific settings

  • Statistical methods applied to antibody-derived data

• Images and blots:

  • Uncropped blot images in supplementary materials

  • Molecular weight markers clearly indicated

  • Representative images alongside quantification

  • Clear explanation of any image processing applied

Adherence to these documentation standards will facilitate replication of findings and advance collective understanding of MEX3B biology.

What are the most promising future directions for MEX3B antibody development and application?

The field of MEX3B research would benefit from several strategic developments in antibody technology:

• Domain-specific monoclonal antibodies: Development of antibodies specifically targeting the KH domains versus the RING finger domain would help dissect the distinct functions of these regions in MEX3B activity.

• Conformation-sensitive antibodies: Antibodies that can distinguish between different conformational states of MEX3B (e.g., RNA-bound versus unbound) would provide insights into activation mechanisms.

• Phospho-specific antibodies: Given the likely role of phosphorylation in regulating MEX3B function, phospho-specific antibodies targeting key regulatory sites would be valuable for studying activation dynamics.

• Intrabodies for live-cell imaging: Cell-permeable antibody fragments or nanobodies that can detect MEX3B in living cells would enable real-time tracking of MEX3B localization and interactions.

• Antibody-based proximity sensors: Development of split fluorescent protein systems fused to anti-MEX3B antibody fragments would allow visualization of protein-protein interactions in live cells.

• Therapeutic antibodies modulating MEX3B function: Given MEX3B's role in TLR3 signaling , antibodies that can selectively enhance or inhibit its function could have therapeutic potential in infectious or inflammatory diseases.

• Degrader antibody conjugates: Antibody-based targeted protein degradation approaches could provide new tools for studying MEX3B function through acute protein depletion.