PHOX2A Antibody

What is PHOX2A and why is it important in neurodevelopmental research?

PHOX2A (paired-like homeobox 2a) is a transcription factor critically involved in neuronal development and differentiation. It plays a significant role in the formation of the anterolateral system (ALS) neurons, which are involved in pain and temperature sensation pathways . PHOX2A is particularly important in neurodevelopmental research because it defines specific neuronal populations during development, and its expression patterns help identify distinct neuronal subtypes . Recent deep sequencing studies have revealed that PHOX2A marks at least five distinct classes of anterolateral system neurons, making it a valuable marker for studying neuronal development and circuit formation . Additionally, PHOX2A has been implicated in neuroblastoma pathogenesis, with alterations in its expression associated with tumor development and progression .

What are the recommended applications for PHOX2A antibody?

Based on validated testing, PHOX2A antibody (such as 25804-1-AP) is primarily recommended for Western Blot (WB) and ELISA applications . For Western blotting, the recommended dilution range is 1:500-1:1000, though researchers should optimize the dilution for their specific experimental system . The antibody has demonstrated reactivity with human samples and can detect PHOX2A protein in cell lines such as SH-SY5Y and HeLa cells . When using the antibody for the first time in a new experimental system, it is advisable to perform a dilution series to determine optimal concentration and validate specificity through appropriate controls, including positive control samples known to express PHOX2A.

What is the molecular weight of PHOX2A and why might observed weights vary?

The calculated molecular weight of PHOX2A is approximately 30 kDa (corresponding to its 284 amino acid sequence), but the observed molecular weight in Western blot applications typically ranges between 35-40 kDa . This discrepancy between calculated and observed molecular weight is common for many proteins and can be attributed to several factors, including post-translational modifications (such as phosphorylation, glycosylation, or ubiquitination), the presence of charged amino acids affecting migration, or protein-specific conformational characteristics that influence mobility during electrophoresis. When analyzing Western blot results, researchers should expect to observe PHOX2A bands in this 35-40 kDa range rather than precisely at the calculated 30 kDa position.

How should PHOX2A antibody be stored to maintain its activity?

For optimal preservation of activity, PHOX2A antibody should be stored at -20°C in its appropriate storage buffer, which typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibody remains stable for one year after shipment. Importantly, aliquoting is generally unnecessary for -20°C storage of this antibody, which simplifies handling procedures . For smaller sized preparations (20μl), the formulation may contain 0.1% BSA as a stabilizing agent. When working with the antibody, it should be thawed completely before use, mixed gently (avoiding vortexing which can damage antibody structure), and kept cold during experimental procedures to maintain its binding capacity.

What is the recommended protocol for detecting PHOX2A expression using Western blot?

For optimal detection of PHOX2A using Western blot, researchers should follow these methodological guidelines: Begin by preparing protein samples from relevant tissues or cell lines (SH-SY5Y and HeLa cells are positive controls) . Separate proteins using 10% SDS-PAGE, then transfer to a nitrocellulose membrane . Block the membrane with an appropriate blocking buffer, then incubate with PHOX2A primary antibody at a 1:500-1:1000 dilution . After washing, incubate with a compatible horseradish peroxidase-conjugated secondary antibody (appropriate for the host species of the primary antibody, typically rabbit for polyclonal PHOX2A antibodies) . Visualize bands using enhanced chemiluminescence detection systems such as Super Signal West Dura . When analyzing results, expect to observe PHOX2A protein bands between 35-40 kDa, despite its calculated molecular weight of 30 kDa . Include molecular weight standards and appropriate controls, including positive control lysates and loading controls such as β-tubulin .

How can researchers isolate PHOX2A-expressing cells or nuclei for downstream applications?

For isolation of PHOX2A-expressing cells or nuclei, researchers have successfully employed transgenic mouse models combined with fluorescence-activated cell sorting (FACS). A validated approach involves using Phox2a::Cre mice crossed with reporter lines such as Sun1-GFP or Ai9(RCL-tdT) to enable visualization of Phox2a-lineage cells . For nuclei isolation specifically, a hypotonic cell lysis protocol followed by FACS has proven effective . In this method, frozen spinal cord tissue is processed to isolate nuclei, which are stained with DRAQ7 to distinguish nuclei from debris . GFP-positive nuclei (representing PHOX2A-expressing cells) can then be sorted, although researchers should note that these cells are relatively rare, typically representing <0.2% of all nuclear events passing through the sorter . This isolation technique enables subsequent applications including single-nucleus RNA sequencing using protocols such as Smart-seq2, allowing for deep transcriptomic profiling of these specific neuronal populations .

What methods can be used to study PHOX2A protein-DNA interactions?

To investigate PHOX2A protein-DNA interactions, chromatin immunoprecipitation (ChIP) has been effectively employed. A validated protocol involves cross-linking proteins to DNA with formaldehyde, followed by chromatin isolation and fragmentation . The chromatin is then incubated overnight at 4°C with anti-PHOX2A antibody (typically 5 μg) with appropriate pre-immune IgY as negative control . Immunocomplexes are collected using either monoclonal anti-chicken IgY-agarose beads or protein G/agarose bead slurry pre-adsorbed with tRNA and salmon sperm DNA to reduce non-specific binding . After washing and elution, cross-linking is reversed by heating to 65°C overnight, followed by DNA purification on columns . PCR detection of the immunoprecipitated chromatin typically uses 5% of the purified DNA as template with specific primers for the target promoter region . This technique allows researchers to identify genomic regions bound by PHOX2A, providing insight into its role as a transcription factor and regulatory networks it controls.

How can researchers quantify PHOX2A gene expression levels?

For accurate quantification of PHOX2A gene expression, researchers should implement quantitative real-time PCR (qRT-PCR) following total RNA extraction and reverse transcription. A validated protocol involves using TaqMan® primer and probe assays specific for PHOX2A (ID #Hs00605931_mH) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ID# Hs99999905_m1) as an endogenous control . Results can be calculated using either the 2−ΔCT method for direct comparisons of PHOX2A expression between samples, or the 2−ΔΔCT method when normalizing to both the endogenous control and a calibrator sample . This approach enables precise measurement of PHOX2A mRNA levels across different experimental conditions, such as during retinoic acid-induced differentiation of neuroblastoma cells. Researchers should verify the compatibility of their endogenous control with their experimental system and include appropriate technical and biological replicates to ensure statistical validity of their findings.

How do PHOX2A and PHOX2B expression patterns differ during retinoic acid-induced differentiation?

PHOX2A and PHOX2B exhibit distinct and opposing expression patterns during retinoic acid-induced differentiation, despite their structural similarities. Research has demonstrated that all-trans retinoic acid (ATRA) treatment leads to the up-regulation of PHOX2A mRNA while simultaneously down-regulating PHOX2B at the transcriptional level . Interestingly, with prolonged ATRA treatment, PHOX2A protein undergoes selective degradation despite its mRNA remaining up-regulated, revealing a complex post-transcriptional regulatory mechanism . This differential regulation suggests distinct roles for these transcription factors during neuronal differentiation. Furthermore, evidence indicates that PHOX2A is capable of modulating PHOX2B expression, though this mechanism is not involved in the ATRA-induced down-regulation of PHOX2B . These complex regulatory interactions highlight the importance of examining both mRNA and protein levels when studying these transcription factors, as transcriptional and post-transcriptional mechanisms may lead to divergent expression patterns, particularly in the context of differentiation-inducing treatments.

How can researchers distinguish between the five classes of PHOX2A-expressing anterolateral system neurons?

Recent deep sequencing of Phox2a nuclei has revealed five distinct classes of anterolateral system (ALS) neurons, requiring sophisticated techniques for their identification and characterization . To distinguish between these neuronal subclasses, researchers should employ a combination of approaches. Single-nucleus RNA sequencing of PHOX2A-GFP positive nuclei isolated by FACS provides the most comprehensive differentiation, allowing identification of specific marker genes for each subclass . For validation and visualization of these distinct populations, multiplex in situ hybridization using RNAscope technology with probes for subclass-specific markers (such as Lypd1, Tacr1, and Tac1) combined with Phox2a lineage tracing is effective . The ALS2 subclass, for example, can be identified by Baiap3 expression and shows significantly higher responses to noxious stimuli as measured by Fos expression . Researchers should note that quality control is essential, typically discarding data from nuclei with fewer than 5,000 unique genes detected and removing non-neuronal contaminating cells based on marker gene expression . This multi-modal approach enables precise classification of these neuronal subtypes, facilitating investigation of their specific roles in sensory processing and response to stimuli.

What are the challenges in detecting post-translational modifications of PHOX2A protein?

Detecting post-translational modifications (PTMs) of PHOX2A presents several methodological challenges that researchers must address. The discrepancy between calculated (30 kDa) and observed (35-40 kDa) molecular weights suggests the presence of PTMs that affect protein mobility during electrophoresis . A particular challenge involves the selective degradation of PHOX2A protein during retinoic acid treatment despite continued mRNA expression, indicating complex post-translational regulation possibly involving the ubiquitin-proteasome pathway . To investigate this, researchers have successfully employed co-immunoprecipitation techniques using anti-PHOX2A antibodies followed by Western blotting with anti-ubiquitin antibodies . This approach requires careful optimization of immunoprecipitation conditions, including the use of bridging antibodies (such as rabbit anti-chicken IgG) when working with chicken anti-PHOX2A antibodies due to their poor binding to protein G . Other potential PTMs, such as phosphorylation, glycosylation, or acetylation, may require specialized techniques including phospho-specific antibodies, glycosidase treatments, or mass spectrometry-based approaches. Researchers should also consider the use of proteasome inhibitors or phosphatase inhibitors during protein extraction to preserve transient modifications that may be rapidly removed or degraded during sample processing.

What are common issues encountered when using PHOX2A antibody for Western blot and how can they be resolved?

Researchers working with PHOX2A antibody in Western blot applications may encounter several common issues that can be systematically addressed. One frequent problem is weak or absent signal, which may be resolved by increasing antibody concentration (starting with 1:500 dilution and adjusting as needed) , extending primary antibody incubation time (overnight at 4°C), or using more sensitive detection systems like enhanced chemiluminescence. Another challenge is the presence of multiple bands or high background, which can be mitigated by increasing blocking stringency, optimizing washing steps, or using antibodies pre-adsorbed against potential cross-reactive species. When the observed molecular weight differs from expectations, researchers should note that PHOX2A typically appears at 35-40 kDa rather than its calculated 30 kDa weight , and variations might reflect tissue-specific post-translational modifications or alternative isoforms. Degradation products can be minimized by using fresh samples and including protease inhibitors during extraction. For challenging samples with low PHOX2A expression, enrichment techniques such as immunoprecipitation prior to Western blotting may be beneficial. Additionally, including appropriate positive controls (such as SH-SY5Y or HeLa cell lysates) and negative controls (tissues or cells known not to express PHOX2A) is essential for validating results .

How can researchers validate the specificity of PHOX2A antibody?

Validating PHOX2A antibody specificity is crucial for ensuring reliable research results. A comprehensive validation approach should include multiple complementary strategies. First, researchers should perform Western blot analysis using positive control samples (such as SH-SY5Y or HeLa cells) to confirm detection of a band at the expected molecular weight range (35-40 kDa). Specificity can be further verified by comparing patterns across tissues with known differential expression of PHOX2A. For definitive validation, genetic approaches such as using samples from Phox2a knockout models or PHOX2A-depleted cells (via siRNA or CRISPR-Cas9) should demonstrate absence or reduction of the specific band. Peptide competition assays, where the antibody is pre-incubated with excess PHOX2A antigen peptide before application to the blot, should abolish specific binding if the antibody is truly specific. Immunoprecipitation followed by mass spectrometry analysis of the pulled-down proteins can confirm that PHOX2A is indeed the major protein being recognized. Additionally, correlation between protein detection by the antibody and mRNA expression measured by qRT-PCR across different samples provides further evidence of specificity . For immunohistochemistry applications, co-localization of antibody staining with in situ hybridization for PHOX2A mRNA offers powerful validation of antibody specificity in tissue contexts .

What are the key considerations when designing experiments to study PHOX2A expression changes during neuronal differentiation?

When designing experiments to study PHOX2A expression changes during neuronal differentiation, researchers should consider several critical factors to ensure robust and interpretable results. First, temporal dynamics are essential—PHOX2A expression should be measured at multiple time points throughout the differentiation process, as research has shown complex regulation patterns including initial upregulation followed by protein degradation during retinoic acid-induced differentiation . Both mRNA and protein levels should be assessed in parallel using qRT-PCR and Western blotting respectively, as post-transcriptional regulation can lead to discordant patterns between transcript and protein abundance . Appropriate cell models should be selected, with neuroblastoma cell lines like SH-SY5Y providing relevant systems for studying neuronal differentiation . When inducing differentiation with agents such as retinoic acid, dose-response relationships should be established, and potential confounding effects of the vehicle (e.g., DMSO) controlled for. Important controls include undifferentiated cells maintained throughout the experiment and possibly differentiation induced by alternative pathways. For mechanistic studies, pharmacological inhibitors of specific pathways (such as proteasome inhibitors to investigate protein degradation) can provide insights into regulatory mechanisms . Co-expression analysis with PHOX2B and other neuronal markers helps contextualize PHOX2A changes within broader differentiation programs. Finally, functional assays measuring neuronal characteristics (neurite outgrowth, electrophysiological properties, etc.) should be correlated with PHOX2A expression changes to establish biological significance.

What challenges exist in isolating PHOX2A-expressing neurons and how can they be overcome?

Isolating PHOX2A-expressing neurons presents several technical challenges that require specific methodological approaches. A primary difficulty is their rarity—these cells typically represent <0.2% of all events during FACS sorting , necessitating processing of large amounts of starting material. To overcome this challenge, researchers have successfully employed transgenic approaches using Phox2a::Cre mice crossed with fluorescent reporter lines (such as Sun1-GFP or Ai9(RCL-tdT)) , enabling visual identification of these neurons. For nuclei isolation specifically, hypotonic lysis protocols followed by FACS have proven effective, with DRAQ7 staining helping distinguish nuclei from cellular debris . A critical quality control consideration is ensuring the purity of isolated populations—researchers should implement stringent gating strategies during FACS and subsequently verify neuronal identity through expression analysis of markers such as Meg3 and Slc17a6, while confirming the absence of inhibitory neuronal markers like Slc32a1 and Gad1 . When working with tissue samples, the anatomical specificity of dissection is crucial, as PHOX2A-expressing neurons have specific distributions in regions such as the superficial and deep dorsal horn . For downstream applications like RNA sequencing, maintaining RNA integrity during isolation is essential, requiring rapid processing and appropriate RNase inhibitors. Single-nucleus approaches have advantages over whole-cell methods for post-mortem or frozen tissues, allowing research on archived samples . Balancing experimental design to include both sexes and different experimental conditions on each plate can help control for technical variables .

How might spatial transcriptomics enhance our understanding of PHOX2A-expressing neuronal populations?

Spatial transcriptomics represents a promising frontier for advancing our understanding of PHOX2A-expressing neuronal populations by preserving critical spatial information that is lost in traditional sequencing approaches. While current deep sequencing of Phox2a nuclei has revealed five distinct classes of anterolateral system neurons , these methods require dissociation and isolation of nuclei, severing the spatial relationships between cell types. Spatial transcriptomics technologies would allow researchers to map the precise anatomical distribution of these neuronal subclasses within intact tissue, revealing potential organizational principles and functional domains. This approach could help determine whether the five identified PHOX2A-expressing neuronal subtypes occupy distinct anatomical niches or form specific spatial arrangements relative to each other and surrounding neural circuits. Additionally, spatial methods could reveal local microenvironmental factors that influence PHOX2A expression and function, potentially identifying regionalized signaling mechanisms that regulate PHOX2A-expressing neurons. By combining spatial transcriptomics with lineage tracing in Phox2a::Cre mice crossed with reporter lines , researchers could track developmental trajectories of these neurons while maintaining information about their final anatomical positions. This could provide crucial insights into how developmental expression of PHOX2A relates to ultimate neuronal identity and circuit integration, potentially revealing new therapeutic targets for neurological disorders associated with alterations in these neuronal populations.

What potential applications exist for PHOX2A antibody in clinical research and diagnostics?

PHOX2A antibody holds significant potential for clinical research and diagnostics, particularly in the context of neuroblastoma (NB) and potentially other neurological disorders. Research has demonstrated that PHOX2A expression is altered in neuroblastoma, with reduced expression in unfavorable tumors and localization near deletion breakpoints in 11q-deleted specimens . This suggests that PHOX2A antibody could be developed as a prognostic marker for neuroblastoma, potentially helping to stratify patients and inform treatment decisions. The finding that both PHOX2A and PHOX2B expression are regulated during retinoic acid treatment, a therapy used for neuroblastoma, further indicates potential utility in monitoring treatment response . Additionally, the specific expression of PHOX2A in anterolateral system neurons involved in pain pathways suggests applications in pain disorder diagnostics and research. Development of standardized immunohistochemical protocols using PHOX2A antibody could enable assessment of neuronal populations affected in neuropathic pain conditions. For research applications, PHOX2A antibody could facilitate investigations into neurodevelopmental disorders, given the protein's role in neuronal differentiation. Future clinical applications might include development of PHOX2A-targeted therapeutics, particularly if specific pathogenic mechanisms involving PHOX2A deregulation are identified. As with any clinical biomarker development, extensive validation would be required, including correlation with clinical outcomes, standardization of detection protocols, and establishment of reference ranges across diverse patient populations.

How might single-cell proteomics complement transcriptomic studies of PHOX2A-expressing neurons?

Single-cell proteomics represents a powerful complementary approach to current transcriptomic studies of PHOX2A-expressing neurons, potentially resolving discrepancies observed between mRNA and protein levels. Current research has revealed that PHOX2A protein can be selectively degraded while mRNA remains upregulated during retinoic acid treatment , highlighting the limitations of transcriptome-only analyses. Single-cell proteomics would enable direct measurement of PHOX2A protein levels and post-translational modifications at the individual cell level, providing a more accurate picture of functional protein abundance. This approach could reveal heterogeneity in protein expression and modification states that might be masked in bulk analyses, potentially identifying additional subtypes beyond the five transcriptomically-defined classes of PHOX2A-expressing neurons . Furthermore, simultaneous measurement of multiple proteins would allow mapping of protein-protein interaction networks within PHOX2A-expressing cells, illuminating the functional context in which PHOX2A operates. Integration of proteomics with transcriptomics in the same cells (using approaches such as CITE-seq adapted for intracellular proteins) could directly correlate transcript and protein levels, providing insights into post-transcriptional regulatory mechanisms. Technically, such studies would require advances in antibody-based detection methods for fixed and permeabilized cells, potentially employing multiplexed antibody staining combined with imaging mass cytometry or similar approaches. The challenges of protein stability and extraction would need to be addressed with optimized protocols specific for these neuronal populations, potentially building upon the nuclear isolation methods already established .

What role might PHOX2A play in the development of new pain management strategies?

PHOX2A's specific expression in anterolateral system (ALS) neurons, which are critical components of pain and temperature sensation pathways, positions it as a potential target for novel pain management strategies . Recent research has revealed that PHOX2A defines at least five distinct classes of ALS neurons, with the ALS2 subclass (identified by Baiap3 expression) showing particularly strong responses to noxious stimuli as measured by Fos expression . This fine-grained characterization of pain-processing neuronal subtypes could enable highly selective therapeutic targeting, potentially reducing side effects associated with current broad-spectrum analgesics. Loss-of-function studies have demonstrated that Phox2a deficiency impairs ALS neuron innervation of their brain targets , suggesting that modulation of PHOX2A activity could potentially alter pain signal transmission. Future therapeutic approaches might include development of small molecules that modulate PHOX2A transcriptional activity or targeted gene therapy approaches using viral vectors with PHOX2A promoter elements to achieve selective expression in pain-processing neurons. Additionally, the transgenic mouse models developed for studying PHOX2A (such as Phox2a::Cre crossed with reporter lines) provide valuable tools for preclinical testing of pain interventions with readouts specifically focused on these neuronal populations. Collaborative research between pain specialists and developmental neurobiologists focusing on PHOX2A could accelerate progress in this field, potentially leading to novel analgesic approaches targeting specific components of the pain processing circuitry defined by PHOX2A expression.