rfx4 Antibody

What is RFX4 and what tissue distribution patterns are observed?

RFX4 (Regulatory Factor X 4) is a transcription factor containing an RFX-type winged-helix DNA-binding domain and belongs to the RFX family. It may activate transcription by interacting directly with the X-box and is structurally related to regulatory factors X1, X2, X3, and X5 . RFX4 has multiple isoforms with distinct tissue distribution patterns:

• RFX4-A, -B, and -C: Expressed exclusively in testis

• RFX4-D: Expressed specifically in normal brain tissues

• RFX4-E and -F: Expressed in gliomas but not in normal brain tissue

Quantitative real-time RT-PCR analysis has shown high expression in normal testis, with brain tissue showing approximately 1% of the expression level compared to testis. Notably, overexpression is observed in approximately 28% (17 of 61) of gliomas, but expression is absent in other tumor types such as lung, esophageal, stomach, colon, or liver cancers .

What applications are RFX4 antibodies suitable for?

RFX4 antibodies are validated for several research applications:

For optimal results, researchers should titrate the antibody in their specific testing systems as reactivity may be sample-dependent .

How should RFX4 antibodies be properly stored and handled?

Proper storage and handling of RFX4 antibodies is critical for maintaining reactivity:

• Storage temperature: Store at -20°C for long-term preservation

• Buffer composition: Typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: Stable for one year after shipment when properly stored

• Aliquoting: Unnecessary for -20°C storage for small (20μl) sizes that contain 0.1% BSA

• Reconstitution: For lyophilized formats, reconstitute according to manufacturer protocols

How can Western blot protocols be optimized for RFX4 detection?

Optimizing Western blot protocols for RFX4 detection requires attention to several key factors:

• Protein extraction: For brain tissue samples, trichloroacetic acid precipitation can be used to obtain more concentrated preparations, which is critical when detecting the less abundant RFX4-D isoform .

• Gel concentration: Use 7.5-10% SDS-PAGE gels for optimal separation, as RFX4 isoforms have molecular weights around 83-84 kDa .

• Antibody concentration: For most RFX4 antibodies, a dilution of 1:500-1:1000 is recommended for Western blotting applications .

• Detection systems: Alkaline phosphatase-conjugated secondary antibodies (1:3000 dilution) have been successfully used for visualizing RFX4 bands .

• Expected molecular weight: The calculated molecular weight of RFX4 is 83 kDa, while the observed molecular weight in Western blots is approximately 84 kDa .

What is the significance of RFX4-E and -F isoforms in glioma research?

RFX4-E and -F isoforms have significant implications for glioma research:

• Tumor-specific expression: These isoforms are specifically expressed in gliomas but not in normal brain tissue, where only RFX4-D is expressed .

• Immunogenicity: RFX4-E and -F proteins can induce antibody responses in glioma patients. Antibodies against the RFX4-D C-terminus (which is shared with RFX4-E and -F) were detected in 5% (3 of 58) of glioma patients but were absent in healthy donors .

• Diagnostic potential: The tumor-specific expression pattern makes these isoforms potential biomarkers for glioma diagnosis. IHC analysis using the DC28 monoclonal antibody showed positive nuclear staining in glioma cells .

• Correlation with expression levels: Overexpression of RFX4 mRNA was observed in patients who developed antibody responses against RFX4, suggesting a relationship between expression levels and immunogenicity .

How can researchers distinguish between different RFX4 isoforms?

Distinguishing between RFX4 isoforms requires strategic experimental approaches:

• PCR-based detection: Isoform-specific primers can be designed for RT-PCR analysis. For example, in previous research, specific primer sets were used to differentiate between RFX4-A, -B, -C, -D, -E, and -F mRNAs .

• Quantitative analysis: Real-time RT-PCR using primers that target common regions (e.g., spanning an intron to avoid genomic DNA amplification) can quantify total RFX4 expression .

• Protein detection: Western blot analysis using antibodies targeting the C-terminal region can differentiate between isoforms based on molecular weight differences. The DC28 monoclonal antibody, produced against recombinant RFX4-D C-terminus protein, could detect RFX4-A and -C proteins in testis, RFX4-D in brain, and RFX4-E and -F in gliomas .

• Epitope selection: When selecting antibodies, consider the specific amino acid regions targeted. Products are available targeting various epitopes, including:

  • AA 684-720 (C-Term)

  • AA 101-200

  • AA 58-89

  • AA 352-401

  • AA 144-193

  • AA 109-158

  • AA 1-641

  • N-Term regions

What controls should be used when working with RFX4 antibodies?

Appropriate controls are essential for validating RFX4 antibody experiments:

• Tissue controls:

  • Positive controls: Brain tissue for RFX4-D, testis for RFX4-A/B/C, and glioma samples for RFX4-E/F

  • Negative controls: Tissues known not to express RFX4 (e.g., lung, liver, colon)

• Loading controls: Anti-actin antibodies have been successfully used as loading controls in Western blot analyses of RFX4 .

• Antibody controls:

  • For IHC: NS-1 culture supernatant can be used as a negative control

  • For ELISA: Sera from healthy donors should be used to establish baseline values, with positivity defined as an OD value exceeding the mean OD of healthy donor sera by three standard deviations

• Recombinant protein controls: Purified recombinant RFX4 proteins (e.g., N-terminal and C-terminal fragments) can serve as positive controls and for antibody characterization .

How can RFX4 autoantibodies be detected in patient samples?

Detection of RFX4 autoantibodies in patient samples can be accomplished through:

• ELISA protocol:

  • Coat 96-well plates with recombinant RFX4 protein (1 μg/mL) in carbonate buffer (pH 9.6) overnight at 4°C

  • Incubate with patient sera (typically at 1:400 dilution)

  • Use goat anti-human IgG labeled with horseradish peroxidase (100 μL/mL) as the secondary antibody

  • Define positive reactions as OD values exceeding the mean OD of healthy donor sera by three standard deviations

• Western blot verification:

  • Separate glioma tissue extracts by SDS-PAGE

  • Blot onto nitrocellulose membranes

  • Incubate with patient sera (1:100 dilution)

  • Detect bound antibodies using alkaline phosphatase-conjugated secondary antibodies (1:3000)

This approach has successfully identified autoantibodies against RFX4-E and -F proteins in 5% of glioma patients, suggesting potential diagnostic applications .

What species reactivity is expected with different RFX4 antibodies?

RFX4 antibodies show varying species reactivity patterns:

Some antibodies demonstrate remarkably broad cross-species reactivity, particularly those targeting the AA 109-158 region, which shows reactivity across multiple vertebrate species and even in yeast .

How can purification methods affect RFX4 antibody performance?

Different purification methods can influence antibody performance:

• Protein A chromatography: Commonly used for polyclonal antibody purification, this method effectively isolates IgG antibodies based on their affinity for protein A .

• Antigen affinity purification: More specific than protein A methods, this approach yields antibodies with higher target specificity, as seen with products purified through this method .

• Impact on applications: While less purified antibodies may be suitable for applications like Western blotting, highly purified antibodies are often necessary for more sensitive applications like immunohistochemistry or immunoprecipitation.

For recombinant RFX4 protein production, expression in E. coli using histidine-tag-containing vectors with subsequent purification on Ni²⁺-NTA columns has proven effective for generating antigens for antibody production and assay standards .

What is the potential diagnostic significance of RFX4 in glioma patients?

RFX4 shows promising diagnostic potential in glioma research:

• Tumor-specific expression: RFX4-E and -F isoforms are specifically expressed in gliomas but not in normal tissues or other tumor types .

• Incidence of overexpression: Approximately 28% of gliomas show overexpression of RFX4 mRNA compared to normal brain tissue .

• Autoantibody development: Autoantibodies against RFX4 are detectable in 5% of glioma patients (3 of 58), with a higher incidence in ependymal tumors (17%) compared to astrocytic tumors (4%) .

• Specificity: No antibody response against RFX4 was observed in healthy donors, suggesting high specificity for glioma patients .

These findings suggest that both RFX4 isoform expression and autoantibody detection could serve as potential biomarkers for glioma diagnosis, particularly for ependymal tumors.

How should researchers interpret conflicting RFX4 expression data?

When faced with conflicting RFX4 expression data, researchers should consider:

• Isoform specificity: Different detection methods may preferentially detect certain isoforms. For example, antibodies targeting the C-terminus might detect all isoforms, while those targeting specific regions might only detect certain variants .

• Quantitative versus qualitative methods: Real-time RT-PCR offers quantitative assessment of mRNA levels, while Western blot or IHC provides information about protein expression and localization .

• Tissue heterogeneity: Within tumors, expression patterns may vary across different regions, potentially leading to sampling discrepancies .

• Methodology differences: Variations in protein extraction methods, antibody concentrations, or detection systems can affect results. For brain tissue specifically, trichloroacetic acid precipitation has been used to concentrate samples for improved detection .

• Cross-referencing multiple techniques: For conclusive results, researchers should employ multiple detection methods, such as combining RT-PCR, Western blot, and IHC to validate expression patterns .

By considering these factors, researchers can better interpret apparently conflicting data and identify the most reliable findings.

What are the key considerations for designing RFX4 immunohistochemistry experiments?

For successful RFX4 immunohistochemistry experiments, researchers should consider:

What methodologies are available for producing custom RFX4 antibodies?

Researchers interested in producing custom RFX4 antibodies can consider these validated approaches:

• Immunization strategy: A combined DNA and protein immunization approach has proven effective:

  • Initial intramuscular immunization with plasmid DNA encoding full-length RFX4-D (using in vivo electroporation)

  • Booster immunization with recombinant protein fragments (e.g., C-terminal 323-735 amino acids)

• Recombinant protein production: Express RFX4 fragments in E. coli using histidine-tag-containing vectors (e.g., pQE30) with purification on Ni²⁺-NTA columns .

• Hybridoma production: Fusion of spleen cells from immunized mice with NS-1 myeloma cells, followed by selection and cloning using specialized media (e.g., ClonaCell-HY Medium D) .

• Screening: Screen hybridoma supernatants by ELISA and Western blotting to identify clones with desired specificity .

• Purification: Purify monoclonal antibodies using appropriate systems (e.g., MAbTrap kit) for final preparation .