rfx3 Antibody

What is RFX3 and why is it important in research?

RFX3 (Regulatory Factor X3) is a transcription factor that plays critical roles in multiple developmental processes. It is required for ciliogenesis and islet cell differentiation during endocrine pancreas development. RFX3 is essential for the differentiation of nodal monocilia and left-right asymmetry specification during embryogenesis. It regulates the expression of genes involved in ciliary assembly (DYNC2LI1, FOXJ1, BBS4) and ciliary motility (DNAH11, DNAH9, DNAH5). Recent research has revealed its importance in neuronal development, with haploinsufficiency linked to neurodevelopmental disorders including autism spectrum disorder, intellectual disability, and ADHD .

What types of RFX3 antibodies are currently available for research?

Several types of RFX3 antibodies are commercially available with different characteristics:

Which species reactivity has been confirmed for RFX3 antibodies?

Antibody reactivity varies by product. The rabbit polyclonal antibody ab92670 has been confirmed to work with both mouse and human samples . The mouse monoclonal antibody PCRP-RFX3-1D11 has confirmed reactivity with human samples . NBP1-86301 has been validated for human and mouse, with citations supporting mouse reactivity in published literature, and is predicted to react with rat samples based on sequence homology (100% similarity) .

What is the predicted molecular weight of RFX3 protein in Western blot applications?

The RFX3 protein has a predicted molecular weight of approximately 83-84 kDa. Specifically, ab92670 documentation lists a predicted band size of 84 kDa , while PCRP-RFX3-1D11 documentation indicates an antigen molecular weight of 83.53 kDa .

What are the recommended applications for different RFX3 antibodies?

Each RFX3 antibody has been validated for specific applications:

What are the optimal conditions for immunohistochemistry with RFX3 antibodies?

For paraffin-embedded tissue sections, heat-induced epitope retrieval (HIER) at pH 6 is recommended when using NBP1-86301 . For ab92670, a dilution of 1/50 has been validated for IHC-P applications on human colon carcinoma tissue . When performing immunocytochemistry with NBP1-86301, PFA fixation followed by Triton X-100 permeabilization is recommended for optimal results .

What controls should be included when working with RFX3 antibodies?

Proper experimental controls are essential:

• Negative controls: Pre-incubation with the immunizing peptide has been shown to block specific staining with ab92670 in IHC-P, confirming antibody specificity .

• Positive controls: RFX3 overexpression lysate compared to vector-only transfected cells can serve as positive and negative controls, respectively, as demonstrated with NBP1-86301 .

• Knockout validation: RFX3 knockout samples provide the gold standard negative control, as demonstrated in recent research where Western blot analysis confirmed no detectable protein in KO iPSC lines .

How can RFX3 antibodies be used to study neurodevelopmental disorders?

Recent research has established a link between RFX3 haploinsufficiency and neurodevelopmental disorders with features including autism spectrum disorder, intellectual disability, and ADHD . Researchers can use RFX3 antibodies to:

• Quantify RFX3 expression: Western blot analysis with RFX3 antibodies can confirm reduced protein levels in heterozygous models (~50% reduction) and complete absence in knockout models .

• Identify affected cell populations: Immunostaining of brain organoids or tissue sections can reveal cell type-specific expression patterns affected by RFX3 deficiency.

• Study developmental trajectories: Combined with scRNA-seq data, immunostaining can help validate transcriptional changes in specific neuronal populations, such as the altered glutamatergic L-R pairs predominantly originating from ExUL in RFX3-deficient organoids .

What approaches can be used to study RFX3 binding to target genes?

To investigate RFX3's function as a transcription factor:

• CUT&RUN-sequencing: This technique has successfully profiled RFX3 binding genome-wide, identifying 4,240 RFX3-specific binding peaks in neurons .

• Motif analysis: Analysis of RFX3 binding regions revealed the RFX3 motif in 86% of peaks, confirming direct DNA binding .

• Chromatin immunoprecipitation: RFX3 antibodies can be used in ChIP experiments to identify genomic binding sites in different cell types.

• Co-immunoprecipitation: RFX3 antibodies can pull down RFX3 along with interacting proteins to identify binding partners.

How can RFX3 antibodies help investigate dosage-dependent effects in cellular models?

Recent research has demonstrated that synaptic genes are sensitive to RFX3 dosage . Researchers can use RFX3 antibodies to:

• Validate model systems: Western blot analysis with RFX3 antibodies can confirm approximately 50% reduced protein levels in heterozygous models and complete absence in knockout models .

• Correlate protein levels with phenotypes: Quantitative Western blot analysis can correlate RFX3 protein levels with observed cellular phenotypes across wild-type, heterozygous, and knockout models.

• Cell type-specific analysis: Immunostaining can reveal differential effects of RFX3 dosage across cell types, particularly in complex systems like organoids.

How do I troubleshoot weak or absent RFX3 signal in Western blots?

When experiencing difficulties with RFX3 detection:

• Antibody concentration: Adjust antibody concentration within the recommended range (e.g., 0.04-0.4 μg/ml for NBP1-86301 or 1/500 dilution for ab92670 ).

• Loading control: Ensure sufficient protein is loaded (≥10 μg total protein, as used in validated Western blots ).

• Expression level verification: RFX3 expression varies by cell type; confirm expression in your sample type before troubleshooting antibody issues.

• Buffer optimization: Test different blocking agents and washing conditions to reduce background and enhance specific signal.

• Sample preparation: Use fresh samples and appropriate protease inhibitors to prevent degradation of RFX3 protein.

What are the best storage conditions for RFX3 antibodies?

For optimal antibody performance and longevity:

• Short-term storage: Store at 4°C for up to two weeks for immediate use .

• Long-term storage: For extended storage, divide into small aliquots (no less than 20 μl) and freeze at -20°C or -80°C .

• Avoid freeze-thaw cycles: Repeated freezing and thawing can degrade antibody quality and performance .

• Follow manufacturer guidelines: Some products may have specific storage recommendations based on formulation.

How can I distinguish between specific and non-specific binding in RFX3 immunostaining?

To ensure specificity in immunostaining experiments:

• Peptide competition: Pre-incubate antibody with the immunizing peptide as a blocking control, as demonstrated with ab92670 in IHC-P applications .

• Knockout controls: Include RFX3 knockout samples as negative controls when available .

• Subcellular localization: Verify that staining pattern matches expected localization for RFX3 (primarily nuclear for this transcription factor, with possible nucleoplasmic vesicle distribution as shown with NBP1-86301 ).

• Secondary antibody controls: Include samples with secondary antibody only to identify background staining.

What cellular processes and structures are regulated by RFX3?

RFX3 regulates multiple critical developmental processes:

• Ciliogenesis: Required for the biogenesis of motile cilia by governing growth and beating efficiency of motile cells .

• Left-right asymmetry: Essential for nodal monocilia differentiation and left-right asymmetry specification during embryogenesis .

• Pancreatic development: Together with RFX6, participates in the differentiation of 4 of the 5 islet cell types during endocrine pancreas development .

• Neuronal development: Recent research reveals roles in neuronal development and synaptic function, with dosage-sensitive effects on synaptic gene expression .

• Transcriptional regulation: Functions by forming heterodimers with other RFX proteins and binding to X-box motifs in promoters of target genes .

How can RFX3 antibodies be used to investigate cilia-related disorders?

Given RFX3's essential role in ciliogenesis:

• Primary ciliopathies: RFX3 antibodies can help investigate mechanisms underlying conditions like hydrocephalus, situs inversus, and corpus callosum malformations observed in Rfx3 knockout mice .

• Co-localization studies: Dual immunostaining with RFX3 antibodies and cilia markers can reveal regulatory relationships.

• Developmental timing: Temporal analysis of RFX3 expression during development can identify critical windows for cilia formation and function.

• Therapeutic target validation: RFX3 antibodies can help validate the efficacy of potential therapeutics aimed at modulating cilia function in disease models.

What is known about RFX3's role in neurodevelopmental disorders?

Recent research has expanded our understanding of RFX3 in brain development:

• Genetic evidence: Haploinsufficiency of RFX3 leads to neurodevelopmental disorders with features including autism spectrum disorder, intellectual disability, ADHD, and behavioral dysregulation .

• Cortical development: RFX3 deficiency affects glutamatergic neurons, particularly those originating from the upper layers (ExUL) of the cortex .

• Synaptic function: RFX3 regulates genes involved in synaptic function, with dosage-sensitive effects observed in heterozygous and knockout models .

• Direct gene regulation: CUT&RUN-sequencing identified 4,240 RFX3-specific binding sites in the human genome, with the RFX3 motif present in 86% of peaks, demonstrating direct transcriptional regulation .