FOX2 Antibody
Description
Introduction to FOX2 Antibody
FOX2 antibodies are specialized reagents targeting the FOX2 protein, also known as RNA Binding Fox-1 Homolog 2 (RBFOX2) or RNA Binding Motif Protein 9 (RBM9). These antibodies are critical tools for studying FOX2's role in mRNA splicing, synaptic plasticity, and cellular differentiation. FOX2 regulates alternative splicing of pre-mRNA, influencing diverse biological processes, including neurodevelopment and fibrosis .
Target Protein Overview: FOX2/RBM9
FOX2 is a 41 kDa RNA-binding protein localized in the nucleus and cytoplasm. Key features include:
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Function: Mediates alternative splicing, mRNA stability, and transcriptional regulation .
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Expression: Ubiquitous, with high levels in brain, heart, and reproductive tissues .
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Clinical Relevance: Implicated in cancer, fibrosis, and neurodevelopmental disorders .
Applications in Research
FOX2 antibodies are widely used in:
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Western Blot (WB): Detects FOX2 at ~41–50 kDa (varies by isoform) .
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Immunohistochemistry (IHC): Localizes FOX2 in nuclei of Purkinje cells and cancer tissues .
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Immunofluorescence (IF): Visualizes FOX2 in neuronal and epithelial cells .
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Knockdown/Overexpression Studies: Validates FOX2's role in splicing regulation .
Regulation of Fibrosis-Associated Splicing
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Study: FOX2 knockdown in scleroderma fibroblasts reduced LH2(long) mRNA, a fibrosis-associated isoform .
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Mechanism: FOX2 binds UGCAUG motifs upstream of exon 13A in LH2 pre-mRNA to promote inclusion .
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Tools: siRNA and FOX2 antibodies (e.g., ab57154) confirmed splicing modulation .
Role in Embryonic Stem Cell Survival
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CLIP-seq Analysis: FOX2 binds 962 sites in FGFR2 pre-mRNA, regulating exon inclusion critical for stem cell pluripotency .
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Functional Impact: FOX2 depletion triggered apoptosis in human embryonic stem cells .
Neuronal Development
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Expression: FOX2 is enriched in cerebellar Purkinje cells and cortical neurons .
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Validation: Neuromics' CH22130 antibody highlighted FOX2's nuclear localization in mouse brain sections .
Validation and Quality Control
FOX2 antibodies are rigorously validated across platforms:
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Here’s a structured FAQ collection for researchers working with FOX2 (RBFOX2) antibodies, synthesized from academic literature, patent data, and experimental protocols:
What experimental controls are critical for FOX2 RNA splicing studies?
Advanced Considerations:
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Negative controls: Use non-targeting IgG in IP assays to rule out nonspecific RNA binding .
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Splicing reporters: Design minigenes containing UGCAUGU motifs to assess FOX2-dependent exon skipping/inclusion .
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Expression normalization: Co-stain with RNA-binding proteins like MBNL1/2 to control for technical variability .
How to resolve contradictions in FOX2 localization across studies?
Troubleshooting Framework:
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Fixation artifacts: Compare fresh-frozen vs. formaldehyde-fixed samples. Nuclear localization is sensitive to overfixation .
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Antibody lot variability: Validate with multiple lots (e.g., ab264154 lots 1 and 2 showed consistent IP efficiency) .
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Species cross-reactivity: Confirm reactivity using species-specific blocking peptides (tested in mouse/rat/human) .
What methodologies optimize FOX2 detection in low-abundance samples?
Technical Recommendations:
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Signal amplification: Use tyramide-based systems for IHC in tissues with sparse FOX2 expression.
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Pre-absorption: Treat lysates with agarose-bound synthetic peptide (aa 1-50) to reduce background .
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Multiplexing: Pair with ER-alpha antibodies to study coregulatory roles in breast cancer models .
How to design FOX2 functional studies in alternative splicing?
Experimental Workflow:
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RNA-seq: Profile splicing events in FOX2-knockdown vs. wild-type cells.
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Motif analysis: Identify 5'-UGCAUGU-3' enrichment near regulated exons .
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Rescue experiments: Express wild-type/mutant FOX2 (e.g., BRCT domain deletions) to test mechanistic requirements .
Example Splicing Target:
| Gene | Exon | FOX2-Dependent Regulation |
|---|---|---|
| TRA2B | E8 | Inclusion (p = 1.2e⁻⁵) |
| CAPN3 | E15 | Skipping (p = 3.8e⁻⁴) |
What are common pitfalls in FOX2 co-immunoprecipitation assays?
Critical Factors:
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RNase contamination: Always include RNase inhibitors to preserve RNA-protein interactions.
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Crosslinker choice: Compare formaldehyde (broad-range) vs. DSP (membrane-permeable) for interaction stability .
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Buffer stringency: Optimize NaCl concentrations (150–300 mM) to reduce nonspecific binding.
How to contextualize FOX2 expression data across omics datasets?
Integration Strategy:
