Rbfox3 Antibody

What is Rbfox3/NeuN and why is it used as a neuronal marker?

Rbfox3, commonly referred to as NeuN, is one of a family of three mammalian Fox homologues. The name RBFox is an acronym of "Feminizing locus on X," as the Fox protein was initially discovered in C. elegans as a gene involved in sex determination. Rbfox3 is approximately 46 kDa and contains a highly conserved RRM type RNA recognition motif, which consists of a small ~70 amino acid structure with 4 beta strands and two alpha-helices . The protein functions in RNA splicing and is expressed heavily and specifically in neuronal nuclei, making antibodies against it excellent markers for neuronal cells. Importantly, Rbfox3/NeuN antibodies show complete absence of staining in astrocytes and other non-neuronal cells, providing clear differentiation between neurons and glia in various applications .

What are the different isoforms of Rbfox3/NeuN identified in research?

Alternative splicing of the Rbfox3 pre-mRNA leads to the production of four protein isoforms that migrate in the 45–50 kDa range. Mass spectrometry analysis has shown that both bands of the characteristic NeuN doublet include a cassette exon (exon 8), which encodes the C-terminal half of the RRM domain . Close inspection of anti-NeuN westerns reveals that the antibody actually recognizes a "doublet of doublets," where both the 45 and 50 kDa 'bands' actually comprise two individual protein species with slight molecular weight differences . The alternative splicing occurs primarily at exon 12 and exon 15, with the 45 kDa bands representing "-exon 12" variants and the 50 kDa bands representing "+exon 12" variants . Additional alternative splicing can occur at exon 6, which contains two alternative 3′ splice sites just 3 nucleotides apart, creating proteins with or without a single glutamine residue .

What applications are Rbfox3/NeuN antibodies typically used for?

Based on the search results, Rbfox3/NeuN antibodies are primarily used for:

• Western blotting (WB): Typically used at concentrations of 0.125 - 1.0 μg/mL

• Immunohistochemistry on formalin-fixed paraffin-embedded tissue sections (IHC-P): Used at concentrations of 1.0 - 10.0 μg/mL

• Immunohistochemistry on frozen tissue sections (IHC-F): Used at concentrations of 1.0 - 10.0 μg/mL

• Immunofluorescence (IF): Used at dilutions around 1:500

• Slot blotting (SB): Verified for this application by community testing

• Spatial biology applications: Some products have been verified for use with platforms like the NanoString GeoMx® Digital Spatial Profiler

What is the precise epitope recognized by anti-Rbfox3/NeuN antibodies?

The anti-NeuN epitope resides at the extreme N-terminus of Rbfox3. Molecular mapping has shown that the N-terminal 21 amino acids of Rbfox3 are both necessary and sufficient for recognition by anti-NeuN antibodies . Specifically, research has identified that a portion of the 46 amino acid R3hdm2 antibody binding site (ABS) is identical in 8 of 15 amino acids to the N-terminal region of Rbfox3 . Deletion construct experiments demonstrated that Rbfox3 amino acids 5–20 are critical for antibody recognition, and these residues are conserved across species . This knowledge is essential for researchers developing new antibodies or evaluating cross-reactivity.

How does alternative splicing affect Rbfox3/NeuN detection in different neural tissues?

Alternative splicing of Rbfox3 can significantly impact antibody detection patterns. RT-PCR studies have shown that different tissues and developmental stages exhibit varying complements of Rbfox3 transcript variants . In P19 cells induced with retinoic acid, the expression pattern of Rbfox3 variants is similar to that seen in P10 mouse brain, where the majority of transcripts lack exon 12, and both exon 15 variants are present .

Some variants, such as those lacking exon 8 (which creates a 31 amino acid deletion within the RRM domain), have been detected at the mRNA level in mouse retina but do not appear to produce detectable protein in the tissues tested . Researchers should be aware that the "doublet of doublets" pattern seen in Western blots reflects these alternative splicing events, and tissues with different splicing patterns may show altered detection profiles.

How can Rbfox3/NeuN antibodies be used to study pathological conditions affecting neurons?

Rbfox3/NeuN antibodies have proven valuable in studying neurological disorders. For example, research has shown that HIV infection significantly reduces expression of Rbfox3/NeuN mRNA and protein in the central nervous system . Quantitative real-time PCR analyses have demonstrated decreased Rbfox3/NeuN mRNA levels in HIV-infected frontal cortex samples compared to non-infected controls .

Immunohistochemistry studies have revealed that Rbfox3/NeuN expression is reduced in both nuclear and cytoplasmic locations in hippocampus and basal ganglia tissues from HIV-positive individuals . These findings suggest that monitoring changes in Rbfox3/NeuN expression using specific antibodies can provide insights into neuronal damage or dysfunction in various pathological conditions. The antibodies can be used to quantify neuronal loss, assess subcellular protein distribution changes, and evaluate the effects of therapeutic interventions.

What are the optimal conditions for using Rbfox3/NeuN antibodies in immunohistochemistry?

For optimal immunohistochemistry results with Rbfox3/NeuN antibodies, consider the following protocol elements:

• Antigen retrieval methods: For paraffin-embedded tissues (IHC-P), antigen retrieval with either Sodium Citrate or Tris-EDTA (10 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH 9.0) is recommended .

• Antibody concentration:

  • For IHC-P: 1.0 - 10.0 μg/mL

  • For IHC-F: 1.0 - 10.0 μg/mL

• Storage and handling:

  • Store antibody solutions undiluted between 2°C and 8°C

  • For fluorophore-conjugated antibodies, protect from prolonged exposure to light

  • Do not freeze conjugated antibodies

• Titration: It is always recommended to titrate the antibody for optimal performance for each specific application and tissue type .

How can subcellular localization of Rbfox3/NeuN be properly interpreted?

While Rbfox3/NeuN is traditionally considered a nuclear marker, research has shown that it can also be detected in the cytoplasm of some neurons . Immunohistochemistry studies have revealed fluorescence clearly above background levels in the cytoplasm of neurons from both healthy and HIV-infected individuals, although the level of expression in both nuclear and cytoplasmic locations is reduced in neurons from HIV-positive individuals .

When interpreting subcellular localization patterns, researchers should:

• Include appropriate controls to establish baseline expression levels

• Consider that changes in nuclear versus cytoplasmic distribution may reflect alterations in protein function or cellular stress

• Be aware that different isoforms of Rbfox3/NeuN may have different subcellular localizations

• Use high-resolution imaging and z-stack analysis to accurately distinguish between genuine cytoplasmic staining and optical artifacts

What are the key considerations for Western blot analysis of Rbfox3/NeuN?

When performing Western blot analysis of Rbfox3/NeuN, researchers should consider:

• Expected banding pattern: Rbfox3/NeuN typically appears as a doublet at 45-50 kDa, which actually represents a "doublet of doublets" due to alternative splicing at exons 12 and 15 .

• Migration discrepancy: Rbfox3 protein variants often migrate on SDS-PAGE gels at molecular weights slightly greater than their calculated values, which should be taken into account when interpreting results .

• Antibody concentration: The suggested use of Rbfox3/NeuN antibodies for Western blotting is 0.125 - 1.0 μg/mL .

• Sample preparation: Given the nuclear localization of much of the Rbfox3/NeuN protein, effective nuclear protein extraction methods are essential for comprehensive analysis.

• Controls: Include positive controls (e.g., mouse brain lysate) and negative controls (non-neuronal cell lysates) to verify antibody specificity.

How can researchers validate the specificity of Rbfox3/NeuN antibodies?

Validating antibody specificity is crucial for reliable experimental results. For Rbfox3/NeuN antibodies, consider:

• Western blot analysis: Confirms the expected doublet pattern at 45-50 kDa in neuronal samples and absence of bands in non-neuronal samples.

• Immunohistochemistry patterns: Verify strong nuclear and distal cytoplasmic staining in neurons with complete absence of staining in astrocytes and other non-neuronal cells .

• Deletion constructs: For advanced validation, researchers can use deletion constructs like Fox3Δ-myc (lacking Rbfox3 amino acids 5–20) and F20-myc (harboring Rbfox3 amino acids 2–20) to confirm antibody epitope specificity .

• Cross-validation: Compare results using antibodies from different sources or with different epitopes.

• Knockout/knockdown controls: When available, use tissues or cells with Rbfox3/NeuN knockout or knockdown as negative controls.

What factors might cause variable Rbfox3/NeuN detection across different experimental conditions?

Variable detection of Rbfox3/NeuN may result from:

• Alternative splicing: Different neural tissues or developmental stages may express different Rbfox3 isoforms that could affect antibody binding .

• Fixation methods: Over-fixation can mask epitopes, particularly for nuclear proteins.

• Antigen retrieval efficiency: Variations in antigen retrieval methods can significantly impact staining intensity in IHC applications.

• Antibody clones: Different antibody clones may recognize different epitopes or isoforms of Rbfox3/NeuN.

• Pathological conditions: Diseases like HIV infection can reduce Rbfox3/NeuN expression levels .

• Sample preparation: Protein extraction methods, particularly for nuclear proteins, can influence detection efficiency.