RFXANK Antibody

What is RFXANK and why is it important in immunological research?

RFXANK is a regulatory protein containing ankyrin repeats that forms the heterotrimeric RFX complex alongside RFX5 and RFXAP. This complex plays a crucial role in the regulation of MHC class II gene expression, which is essential for antigen presentation to CD4+ T cells and proper immune function. RFXANK is particularly important in immunological research because mutations in this gene account for approximately two-thirds of all cases of MHC class II deficiency, a severe combined immunodeficiency disorder . Research using RFXANK antibodies helps elucidate the molecular mechanisms of MHC class II expression regulation and immune system development, providing insights into both normal immune function and immunodeficiency disorders .

What techniques can RFXANK antibodies be used for in research?

RFXANK antibodies can be employed in multiple research techniques to study protein expression, localization, and function. According to available data, these antibodies are validated for immunohistochemistry (IHC) at dilutions of 1:20-1:50 and immunofluorescence (IF) at concentrations of 0.25-2 μg/mL . Additionally, researchers have successfully used these antibodies in coimmunoprecipitation assays to study protein-protein interactions, particularly for investigating how RFXANK interacts with other components of the RFX complex . Western blotting applications are also common, especially for detecting RFXANK in enhanceosome complexes pulled down in promoter assays . Techniques like chromatin immunoprecipitation (ChIP) can be employed to study the binding of RFXANK to MHC class II promoter regions, though specific optimization may be required.

How should RFXANK antibodies be stored and handled to maintain activity?

For optimal performance and longevity, RFXANK antibodies should be stored at -20°C in their buffer of glycerol solution . When handling the antibody, it is recommended to minimize freeze-thaw cycles by aliquoting the stock solution into smaller volumes upon initial thawing. Working dilutions should be prepared fresh before each experiment and stored at 4°C for no more than 24 hours. During shipping, the antibodies are typically transported on wet ice to maintain stability . For long-term storage beyond one year, some researchers recommend keeping aliquots at -80°C, though manufacturer guidelines should be consulted. When working with the antibody, avoid contamination by using sterile techniques and never introduce foreign substances into the original container.

What are common controls used when working with RFXANK antibodies?

Effective experimental design with RFXANK antibodies requires appropriate controls to validate specificity and performance. Positive controls should include cell types or tissues known to express RFXANK, such as professional antigen-presenting cells (dendritic cells, B cells, macrophages). The complemented BLS-1c cell line (RFXANK-positive) serves as an excellent positive control, while the RFXANK-deficient BLS-1 cell line provides a matched negative control . For immunohistochemistry or immunofluorescence, researchers should include isotype controls to account for non-specific binding. Peptide blocking experiments using the specific immunogen sequence (TQPAEDLIQTQQTPASELGDPEDPGEEAADGSDTVVLSLFPCTPEPVNPEPDASVSSPQ) can confirm antibody specificity . For Western blotting or immunoprecipitation, lysates from cells transfected with RFXANK expression vectors provide useful positive controls, while knockdown or knockout systems offer negative controls to verify specificity.

How can RFXANK antibodies be used to study the RFX complex assembly and function?

RFXANK antibodies provide powerful tools for dissecting the intricate formation and function of the RFX complex. Researchers can employ coimmunoprecipitation assays with anti-RFXANK antibodies to pull down the entire complex and analyze protein-protein interactions with RFX5 and RFXAP . For more detailed mechanistic studies, coupling RFXANK antibodies with site-directed mutagenesis approaches allows mapping of specific interaction domains. This methodology was successfully used to identify that the RFXANK-RFX5 interaction domain maps to an outer surface of ankyrin repeats 2 and 3 . For studying the dynamics of complex assembly, sequential immunoprecipitation can be performed using antibodies against different complex components. Chromatin immunoprecipitation (ChIP) assays using RFXANK antibodies, followed by sequencing (ChIP-seq), can map genome-wide binding profiles to identify all target genes regulated by the RFX complex beyond the well-characterized MHC class II loci.

How can RFXANK antibodies be employed in deciphering the regulatory networks of MHC class II expression?

RFXANK antibodies serve as critical tools for mapping the complex regulatory networks controlling MHC class II expression. Researchers can implement ChIP-seq approaches with RFXANK antibodies to identify genome-wide binding sites and potential non-canonical target genes beyond the classical MHC loci. Sequential ChIP (re-ChIP) using antibodies against RFXANK followed by other transcription factors can reveal co-occupancy patterns and identify cooperative binding relationships . For studying dynamic regulation, ChIP combined with kinetic analyses following immune stimulation can track temporal changes in RFXANK binding. Proximity ligation assays (PLA) using RFXANK antibodies paired with antibodies against other chromatin-associated factors can visualize protein-protein interactions in situ. Additionally, proteomics approaches like immunoprecipitation followed by mass spectrometry (IP-MS) can identify novel RFXANK-interacting proteins that may represent undiscovered components of the regulatory network. The promoter pull-down assay developed for assessing enhanceosome assembly can be adapted with RFXANK antibodies to study how different stimuli affect complex formation on MHC class II promoters .

What methodological adaptations are needed when using RFXANK antibodies in different cell types?

Using RFXANK antibodies across different cell types requires specific methodological adaptations to ensure reliable results. For primary human dendritic cells and macrophages, which naturally express high levels of RFXANK, lower antibody concentrations (approximately 0.25-0.5 μg/mL for immunofluorescence) may be sufficient, while B-cell lines might require the higher end of the recommended range (1-2 μg/mL) . Cell-type specific fixation protocols are critical: lymphoid cells typically require gentler fixation (2% paraformaldehyde for 10 minutes), while adherent cells may need stronger conditions (4% paraformaldehyde for 15-20 minutes). When working with tissue sections, antigen retrieval methods should be optimized - epitope masking is common in formalin-fixed tissues and may require citrate buffer (pH 6.0) heat-induced retrieval. For flow cytometry applications in mixed cell populations, combination with lineage markers is essential to distinguish cell-type specific expression patterns. Researchers working with model systems should note that while RFXANK antibodies are often raised against human proteins, cross-reactivity with other species must be empirically determined and validated for each application .

How should experiments be designed to study RFXANK mutations using antibodies?

Designing experiments to study RFXANK mutations requires a comprehensive approach combining antibody-based detection with functional assays. Begin by selecting antibodies that recognize epitopes outside the mutated region to ensure the mutant protein can still be detected. For studying the well-documented 26-bp deletion mutation (I5E6-25_I5E6+1) that accounts for a significant portion of MHC class II deficiency cases in North Africa, antibodies recognizing the N-terminal region would be appropriate . Design complementation assays using RFXANK-deficient cell lines (such as BLS-1) transfected with various mutant constructs, then evaluate MHC class II restoration using flow cytometry alongside RFXANK detection via immunoblotting . For analyzing protein-protein interactions, employ coimmunoprecipitation with antibodies against RFX5 or RFXAP partners and probe for mutant RFXANK . Create chimeric constructs replacing specific ankyrin repeats with those from related proteins (like ANKRA2) to map functional domains precisely. Use subcellular fractionation followed by immunoblotting to assess whether mutations affect nuclear localization. For longitudinal studies of mutation effects, establish inducible expression systems where mutant RFXANK can be conditionally expressed while monitoring downstream gene expression changes.

What are common troubleshooting strategies for weak or non-specific RFXANK antibody signals?

When encountering weak or non-specific signals with RFXANK antibodies, several troubleshooting approaches can yield improvements. For weak signals in immunoblotting, first optimize protein extraction methods to ensure complete lysis - the RFXANK protein may be tightly associated with nuclear components requiring more stringent extraction buffers containing 0.5-1% NP-40 . Increase antibody concentration incrementally (up to 2-fold) while extending primary antibody incubation time to overnight at 4°C. For immunohistochemistry with weak signals, try alternative antigen retrieval methods or increase retrieval time. Non-specific signals can be addressed by implementing more stringent blocking (5% BSA instead of standard blocking reagents) and adding 0.1-0.3% Triton X-100 to reduce background. In coimmunoprecipitation experiments with high background, increase the number and duration of washes, and consider using more stringent washing buffers containing up to 250-300 mM NaCl . If nuclear localization signals are difficult to detect, add a nuclear stain (DAPI) and optimize confocal microscopy settings to enhance nuclear detail. For flow cytometry applications with high background, implement tighter gating strategies and include dead cell exclusion dyes to eliminate autofluorescent dead cells.

How can RFXANK antibodies be optimized for chromatin immunoprecipitation studies?

Optimizing RFXANK antibodies for chromatin immunoprecipitation requires specific protocol adaptations to ensure efficient chromatin binding and precipitation. Begin with antibody selection - choose antibodies validated for ChIP or those recognizing epitopes likely to remain accessible when RFXANK is bound to DNA (avoid antibodies targeting DNA-binding domains). Perform antibody validation using ChIP-qPCR on known RFXANK binding sites in the HLA-DRA promoter region before proceeding to genome-wide studies . For crosslinking optimization, test both formaldehyde concentrations (0.5-1.5%) and crosslinking times (5-15 minutes) as excessive crosslinking can mask epitopes. Sonication conditions require careful calibration - aim for chromatin fragments between 200-500bp for optimal resolution, verifying fragmentation by agarose gel electrophoresis. Pre-clear chromatin samples thoroughly with protein A/G beads before adding the RFXANK antibody to reduce background. For antibody concentration, begin with 2-5 μg per ChIP reaction and adjust based on preliminary results. Include IgG controls matched to the RFXANK antibody species and isotype. For washing steps, implement a gradient of increasingly stringent wash buffers to remove non-specific interactions while preserving specific RFXANK-DNA complexes. Consider using dual crosslinking with both formaldehyde and protein-specific crosslinkers to stabilize protein-protein interactions within the RFX complex.

What quantitative methods provide the most reliable results when using RFXANK antibodies?

For generating reliable quantitative data with RFXANK antibodies, several methodological approaches offer particular advantages. Quantitative Western blotting provides highly reproducible results when implemented with fluorescently-labeled secondary antibodies and digital imaging systems rather than chemiluminescence, allowing precise quantification within a broader linear range . Include loading controls targeting housekeeping proteins from the same subcellular compartment as RFXANK (nuclear proteins like Lamin B). For flow cytometry, implement a quantitative fluorescence calibration using standardized beads to convert arbitrary fluorescence units to absolute antibody binding capacity. In immunofluorescence quantification, utilize automated image analysis software with consistent thresholding parameters across all samples, and report intensity as a ratio to nuclear staining to normalize for cell-to-cell variations. For ChIP-qPCR quantification, the percent input method generally provides more reliable results than fold enrichment calculations, particularly when comparing samples with variable backgrounds. When conducting coimmunoprecipitation experiments, quantify co-precipitated proteins as a ratio to the immunoprecipitated RFXANK to control for precipitation efficiency variations . For absolute quantification, develop a standard curve using recombinant RFXANK protein at known concentrations. In all cases, biological replicates (n≥3) are essential for statistical validation, and technical replicates help identify and mitigate methodological variability.

How do RFXANK expression patterns correlate with MHC class II expression in different immune cell populations?

RFXANK expression patterns exhibit specific correlations with MHC class II expression that vary among immune cell populations. Professional antigen-presenting cells (dendritic cells, B cells, macrophages) show constitutive expression of both RFXANK and MHC class II molecules, with a positive correlation between their expression levels . In activated B cells, RFXANK protein levels increase within 12-24 hours following activation stimuli, preceding the upregulation of MHC class II surface expression by approximately 6-12 hours, suggesting a causal relationship. Dendritic cells maintain high baseline expression of RFXANK, which correlates with their constitutively high MHC class II expression. Non-professional antigen-presenting cells, including thymic epithelial cells, show coordinate expression of RFXANK and MHC class II, crucial for T cell development. In contrast, cells that do not normally express MHC class II (like T cells, NK cells, and most non-hematopoietic cells) express RFXANK at very low or undetectable levels. Importantly, in MHC class II deficiency patients with RFXANK mutations, cells may express mutant RFXANK protein but completely lack MHC class II expression, demonstrating that protein presence alone is insufficient - functional activity is required . During cellular differentiation of monocytes to macrophages or dendritic cells, RFXANK upregulation precedes MHC class II expression, suggesting its role as an upstream regulator in this developmental pathway.

What insights have RFXANK antibodies provided about the structure-function relationship of the protein?

RFXANK antibodies have been instrumental in elucidating critical structure-function relationships within this regulatory protein. Through antibody-based studies combined with mutagenesis approaches, researchers have mapped specific functional domains within RFXANK's ankyrin repeat domain (ARD) . These studies revealed that the RFXANK-RFX5 interaction domain maps to an unprecedented outer surface of ankyrin repeats 2 and 3, demonstrating a novel interaction surface for ankyrin repeat proteins . Furthermore, antibody studies helped identify that mutations within ankyrin repeats 1 and 3 interfere with the formation of the RFX complex without abolishing RFXANK's ability to bind individually to RFX5 or RFXAP, suggesting a sequential assembly process . The C-terminal region containing the ARD (amino acids 84 to 260) has been definitively established as both essential and sufficient for RFXANK function through complementation studies in RFXANK-deficient cells, detected using specific antibodies . Interestingly, chimeric protein studies using RFXANK antibodies demonstrated that the ARD of the related protein ANKRA2 can functionally replace the ARD of RFXANK, highlighting the structural conservation of these domains despite different cellular functions . Through precise mapping with antibodies, researchers determined that while the first 83 amino acids of RFXANK are dispensable for function, the inclusion of N-terminal regions enhances protein stability and complex formation efficiency.

How can RFXANK antibody data be integrated with genetic and clinical findings in immunodeficiency research?

Integrating RFXANK antibody data with genetic and clinical findings requires a multidisciplinary approach that connects molecular mechanisms to disease manifestations. When analyzing patient samples with suspected MHC class II deficiency, combine genomic sequencing to identify RFXANK mutations with antibody-based detection of protein expression and functional assessment of MHC class II surface expression . This integrated approach can distinguish between mutations affecting protein expression versus those producing non-functional proteins. In founder mutation studies, such as the 26-bp deletion prevalent in North African populations, antibody detection of RFXANK in carrier individuals (heterozygotes) can assess whether protein expression levels correlate with subtle immunological phenotypes not reaching clinical threshold . For genotype-phenotype correlations, classify patients based on specific RFXANK mutation types and correlate with antibody-detected protein levels, MHC class II expression, CD4+ T cell counts, and clinical severity metrics . In therapeutic monitoring of hematopoietic stem cell transplantation (HSCT) patients, sequential analysis of RFXANK and MHC class II expression in immune cells can track reconstitution of the antigen presentation pathway . When investigating novel RFXANK variants of uncertain significance, combine in silico pathogenicity predictions with functional studies using antibodies to detect protein expression, localization, and complex formation capabilities. This integrated approach provides a comprehensive assessment connecting genomic variants to molecular mechanisms and clinical outcomes.

What methodological advances are needed for studying RFXANK in tissue-specific contexts?

Advancing the study of RFXANK in tissue-specific contexts requires several methodological innovations to overcome current limitations. Development of highly specific monoclonal antibodies recognizing different epitopes would enable more precise mapping of protein interactions and conformational changes in different tissues . Multiplexed immunofluorescence techniques combining RFXANK antibodies with tissue-specific markers and other RFX complex components would allow visualization of regulatory networks in their native context. For studying low-abundance expression in non-immune tissues, signal amplification methods like tyramide signal amplification (TSA) or rolling circle amplification should be optimized specifically for RFXANK detection. Tissue-clearing techniques combined with whole-mount immunostaining using RFXANK antibodies would enable three-dimensional visualization of expression patterns across entire organs. For quantitative tissue analysis, mass cytometry (CyTOF) with metal-conjugated RFXANK antibodies would allow simultaneous detection of multiple markers in rare cell populations. Development of proximity ligation assays specific for RFXANK interactions would enable visualization of protein complexes in situ with single-molecule resolution. For functional studies in specific tissues, conditional knockout models with tissue-specific reporters, combined with validated antibodies, would allow tracking of both RFXANK deletion efficiency and consequent changes in MHC class II expression. Finally, the adaptation of single-cell technologies to include RFXANK protein detection alongside transcriptomics would provide unprecedented insights into cell-type specific regulatory mechanisms in complex tissues.