PHOX2A Antibody, Biotin conjugated

What is PHOX2A and why is it important in research?

PHOX2A is a paired-like homeodomain transcription factor that plays essential roles in neuronal differentiation throughout the developing sympathetic, parasympathetic and enteric ganglia. Research significance stems from its critical function in neuronal lineage determination, particularly in the differentiation of the main noradrenergic center of the brain, the locus ceruleus. PHOX2A and its closely related paralog PHOX2B regulate the expression of key enzymes in catecholamine synthesis pathways, including tyrosine hydroxylase and dopamine-beta hydroxylase, which are transiently expressed in neural crest cells . PHOX2A is particularly important in neuroblastoma research, as its expression is finely controlled during retinoic acid-induced differentiation, making it a potential biomarker for staging, prognosis, and treatment decision-making in neuroblastoma .

What are the specific applications of PHOX2A Antibody, Biotin conjugated?

The biotin-conjugated PHOX2A antibody (AA 41-140) is specifically designed for multiple research applications:

• Western Blotting (WB): Optimal dilution range 1:300-5000 for detecting PHOX2A protein expression levels in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative determination of PHOX2A levels

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Dilution range 1:200-400 for visualizing PHOX2A expression in fixed tissues

• Immunohistochemistry on frozen sections (IHC-F): Dilution range 1:100-500 for detecting PHOX2A in cryopreserved tissue samples

The biotin conjugation offers enhanced sensitivity through signal amplification when used with streptavidin detection systems, particularly valuable for detecting low-abundance transcription factors in neural tissues.

How should I store and handle PHOX2A Antibody, Biotin conjugated?

For optimal performance and stability of the PHOX2A Antibody, Biotin conjugated (ABIN1392097):

• Store at -20°C for long-term preservation

• The antibody is supplied in liquid format at 1 μg/μL concentration in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

• Avoid repeated freeze-thaw cycles by aliquoting upon first thaw

• When handling, always wear appropriate personal protective equipment as the preservative ProClin is classified as poisonous and hazardous

• Centrifuge briefly before opening the vial to ensure collection of the entire volume

• Working dilutions should be prepared fresh before use and discarded after the experiment

What are the recommended dilution protocols for different experimental applications?

Always perform optimization experiments to determine the ideal dilution for your specific sample type and application. The biotin conjugation allows for detection using streptavidin-HRP or streptavidin-fluorophore systems, providing flexibility in detection methodologies .

What is the specificity of PHOX2A Antibody, Biotin conjugated?

The PHOX2A Antibody (ABIN1392097) targets amino acids 41-140 of human PHOX2A. It is generated using a KLH-conjugated synthetic peptide derived from human PHOX2A as the immunogen. While primarily validated for human samples, it is predicted to cross-react with PHOX2A from multiple species including mouse, rat, cow, sheep, horse, chicken, and rabbit, making it versatile for comparative studies .

Cross-reactivity with PHOX2B should be carefully assessed since PHOX2A and PHOX2B are closely related paired-homeodomain transcription factors with high sequence homology. Validation experiments including positive and negative controls are essential to confirm specificity in your experimental system.

How can I optimize Western blot protocols when using PHOX2A Antibody, Biotin conjugated?

Optimizing Western blot protocols for PHOX2A detection requires attention to several critical parameters:

• Sample preparation:

  • Use nuclear extraction protocols as PHOX2A is a nuclear transcription factor

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if phosphorylation status is important

• Gel percentage and transfer conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution of PHOX2A (~30-33 kDa)

  • Cold transfer at 30V overnight often yields better results for transcription factors

• Blocking and antibody incubation:

  • Test both milk and BSA as blocking agents (3-5%)

  • For biotin-conjugated antibodies, use avidin/biotin blocking kits to reduce endogenous biotin background

  • Dilute primary antibody in range of 1:300-5000 based on expression level

• Detection system:

  • Use streptavidin-HRP followed by enhanced chemiluminescence

  • Titrate streptavidin-HRP to minimize background while maintaining sensitivity

  • Consider using low-fluorescence PVDF membranes if using streptavidin-fluorophore detection

When analyzing PHOX2A expression patterns during cell differentiation, be aware that retinoic acid treatment can lead to discrepancies between protein and mRNA levels due to post-transcriptional regulation mechanisms .

What are the considerations for using this antibody in immunohistochemistry of neuronal tissues?

When conducting immunohistochemistry on neuronal tissues:

• Antigen retrieval is critical:

  • For paraffin sections: Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • For frozen sections: Brief fixation in 4% paraformaldehyde may improve results

• Tissue specificity considerations:

  • PHOX2A is expressed in specific neuronal populations including the locus coeruleus, parasympathetic ganglia, and enteric nervous system

  • In the retrotrapezoid nucleus (RTN), PHOX2A expression patterns are developmentally regulated and may overlap with PHOX2B

• Signal amplification options:

  • The biotin conjugation allows for detection with streptavidin-based amplification systems

  • ABC (Avidin-Biotin Complex) amplification is recommended for tissues with low expression

  • Tyramide signal amplification (TSA) can further enhance detection of low-abundance targets

• Counterstaining strategy:

  • Nuclear counterstains should be selected to contrast with the chromogen used

  • For co-localization studies with other neuronal markers, carefully select complementary fluorophores that don't bleed into the streptavidin-fluorophore channel

For developmental studies, be aware that PHOX2A expression changes significantly during neuronal differentiation, with cells that begin to differentiate along a neuronal lineage continuing to express PHOX2B and beginning to express PHOX2A .

How can I validate the specificity of PHOX2A Antibody in my experimental system?

Rigorous validation is essential to ensure reliable results:

• Positive and negative controls:

  • Positive control: Tissues/cells known to express PHOX2A (e.g., locus coeruleus, sympathetic ganglia)

  • Negative control: Tissues/cells that don't express PHOX2A (e.g., liver, skeletal muscle)

  • Competitive blocking with immunizing peptide

• Genetic validation approaches:

  • siRNA or CRISPR-mediated PHOX2A knockdown/knockout

  • Overexpression of tagged PHOX2A and co-staining with tag-specific antibody

• Orthogonal method validation:

  • Compare protein detection with mRNA expression (qPCR, in situ hybridization)

  • Use multiple antibodies targeting different epitopes of PHOX2A

  • Western blot validation of IHC results when possible

• Cross-reactivity assessment:

  • Test in systems expressing PHOX2B but not PHOX2A to confirm absence of cross-reactivity

  • Careful comparison with PHOX2B staining patterns in tissues expressing both factors

Given that retinoic acid treatment can lead to different expression patterns of PHOX2A mRNA and protein, validation across multiple techniques is particularly important in differentiation experiments .

How does PHOX2A expression change during retinoic acid-induced differentiation?

PHOX2A undergoes complex regulation during retinoic acid-induced neuronal differentiation:

• Transcriptional upregulation:

  • Treatment with trans-retinoic acid (ATRA) initially upregulates PHOX2A at the mRNA level

  • This upregulation occurs at the transcriptional level and persists throughout treatment

• Post-transcriptional regulation:

  • Despite maintained mRNA upregulation, prolonged ATRA treatment leads to selective degradation of PHOX2A protein

  • This creates a disconnect between mRNA and protein levels that researchers must account for in experimental design

• Contrasting regulation with PHOX2B:

  • While PHOX2A mRNA is upregulated, PHOX2B is downregulated at both mRNA and protein levels

  • This differential regulation suggests distinct roles during neuronal differentiation

• Experimental implications:

  • Time-course experiments should include both protein and mRNA analyses

  • Assessment of PHOX2A as a differentiation marker requires protein-level verification

  • PHOX2A and PHOX2B expression patterns together may serve as useful biomarkers for neuroblastoma staging, prognosis, and treatment decision-making

This fine control of PHOX2A expression during differentiation underscores the importance of temporal considerations in experimental design when studying neuronal development or neuroblastoma differentiation.

What are the best practices for multiplexing PHOX2A antibody with other neural markers?

Effective multiplexing strategies for PHOX2A with other neural markers:

• Antibody selection considerations:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • For biotin-conjugated PHOX2A antibody, avoid other biotin-conjugated antibodies

  • When using multiple rabbit antibodies, consider sequential immunostaining with complete stripping between rounds

• Recommended marker combinations:

  • For sympathetic lineage: Pair with tyrosine hydroxylase (TH) and dopamine-beta hydroxylase (DBH)

  • For central neurons: Combine with NeuN (general neuronal marker) and locus coeruleus markers

  • For developmental studies: Include markers for neural crest cells and precursors

• Detection strategy:

  • Use streptavidin coupled to a far-red fluorophore for PHOX2A detection

  • Select spectrally distinct fluorophores for other markers

  • Carefully test for and eliminate bleed-through using single-marker controls

• Sequential staining protocol:

  • Start with the lowest abundance marker (often PHOX2A)

  • Apply streptavidin detection before introducing other antibodies

  • For chromogenic detection, use dual or triple immunoenzyme labeling with distinct chromogens

Research on CCHS has shown that PHOX2B mutations can affect the expression and localization patterns of both PHOX2A and PHOX2B, with some mutations potentially disrupting the normal nuclear localization of these transcription factors .

How can I use PHOX2A Antibody in research related to congenital central hypoventilation syndrome (CCHS)?

PHOX2A antibody can be valuable in CCHS research contexts:

• Investigation of PHOX2A-PHOX2B interactions:

  • Proximity ligation assays have demonstrated that both wild-type and mutant PHOX2B proteins can interact with PHOX2A

  • The biotin-conjugated antibody can be used in pull-down assays to assess how PHOX2B mutations affect interaction with PHOX2A

• Transcriptional activity assessment:

  • PHOX2B directly regulates the PHOX2A promoter

  • In luciferase reporter assays, mutant PHOX2B shows altered ability to activate the PHOX2A promoter

  • The antibody can help correlate protein levels with transcriptional activity

• Neural population analysis:

  • In the retrotrapezoid nucleus (RTN), PHOX2A-expressing neurons function as central respiratory chemoreceptors

  • PHOX2A antibody can help characterize the development and maintenance of these neurons in CCHS models

  • Co-staining with markers for CO₂ sensing (TASK-2, GPR4) can provide insights into chemoreceptor function

• Protocol for subcellular localization studies:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • Perform antigen retrieval in citrate buffer pH 6.0

  • Use a dilution of 1:200-400 for IHC-P applications

  • Counterstain with DAPI to visualize nuclei

  • Analyze nuclear vs. cytoplasmic staining patterns

CCHS-causing PHOX2B mutations (particularly polyalanine expansions) can lead to protein aggregation and altered subcellular localization, which can be visualized using properly validated PHOX2A and PHOX2B antibodies .

What are common issues when using PHOX2A Antibody, Biotin conjugated and how to resolve them?

When working with retinoic acid-treated samples, remember that PHOX2A protein levels may be significantly reduced despite elevated mRNA levels, potentially giving seemingly contradictory results between protein detection methods and qPCR .

How can I distinguish between PHOX2A and PHOX2B signals in my experiments?

Distinguishing between these closely related transcription factors requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies targeting non-homologous regions of PHOX2A and PHOX2B

  • The PHOX2A antibody targeting AA 41-140 minimizes cross-reactivity with PHOX2B

  • Validate specificity using recombinant proteins or overexpression systems

• Expression pattern differences:

  • PHOX2A expression is more restricted than PHOX2B, with predominant expression in the locus coeruleus

  • PHOX2B is expressed in all neural crest-derived cells initially, while PHOX2A appears during neuronal differentiation

  • During retinoic acid treatment, PHOX2A is upregulated while PHOX2B is downregulated at the mRNA level

• Dual immunostaining approach:

  • Use differently conjugated antibodies (e.g., biotin-PHOX2A and fluorophore-conjugated PHOX2B)

  • Perform sequential staining with complete stripping between rounds

  • Include single-stain controls to ensure specificity

• Molecular techniques for verification:

  • Complement protein detection with mRNA analysis (qPCR with gene-specific primers)

  • Use siRNA/shRNA knockdown of each factor separately to confirm antibody specificity

The differential regulation of PHOX2A and PHOX2B during retinoic acid treatment provides a useful experimental system for validating the specificity of antibodies against these factors .

What controls should I include when using PHOX2A Antibody, Biotin conjugated?

A comprehensive control strategy ensures reliable interpretation of results:

• Positive controls:

  • Locus coeruleus tissue samples or neuronal cell lines known to express PHOX2A

  • Cells transfected with PHOX2A expression vectors

  • Neuroblastoma cells treated with retinoic acid (for early timepoints)

• Negative controls:

  • Primary antibody omission (to detect non-specific binding of detection reagents)

  • Isotype control (rabbit IgG at the same concentration)

  • Tissues or cells known not to express PHOX2A

  • For biotin-conjugated antibodies: streptavidin-only controls to detect endogenous biotin

• Specificity controls:

  • Pre-adsorption of antibody with immunizing peptide

  • PHOX2A knockdown/knockout samples

  • Cells expressing only PHOX2B to check for cross-reactivity

• Technical controls:

  • Loading controls for Western blot (nuclear protein such as Lamin B)

  • Tissue preservation controls for IHC (staining with antibodies to abundant proteins)

  • For multiplex experiments, single-color controls to detect bleed-through

These comprehensive controls are particularly important when studying complex regulatory relationships between PHOX2A and PHOX2B in developmental contexts or disease models .

How can I interpret conflicting results between PHOX2A protein and mRNA expression?

Resolving discrepancies between protein and mRNA data requires understanding potential regulatory mechanisms:

• Post-transcriptional regulation mechanisms:

  • Retinoic acid treatment specifically leads to elevated PHOX2A mRNA but decreased protein levels over time

  • This phenomenon suggests active protein degradation despite continued transcription

  • Similar mechanisms may occur in developmental contexts

• Experimental approaches to resolve discrepancies:

  • Time-course experiments measuring both mRNA and protein levels

  • Proteasome inhibitor treatment to test if protein degradation is occurring

  • Polysome profiling to assess translation efficiency

  • Protein stability assays using cycloheximide chase

• Analytical framework:

  • When mRNA increases but protein decreases: consider enhanced degradation

  • When protein increases but mRNA stable/decreases: consider increased translation efficiency or protein stability

  • When patterns differ between compartments: consider altered subcellular localization

• Biological significance interpretation:

  • Discrepancies often reflect important regulatory mechanisms

  • In neuroblastoma, the PHOX2A mRNA/protein discrepancy during differentiation may serve as a potential biomarker

  • Similar discrepancies may occur in other developmental or disease contexts

Research has shown that in retinoic acid-treated neuroblastoma cells, PHOX2A upregulation at the mRNA level accompanied by subsequent protein degradation is a biologically significant pattern that may correlate with differentiation status .

How should I design experiments to study PHOX2A and PHOX2B interactions?

Studying interactions between these related transcription factors requires specialized approaches:

• Co-immunoprecipitation strategy:

  • Use biotin-conjugated PHOX2A antibody for pull-down with streptavidin beads

  • Probe with PHOX2B antibody to detect interaction

  • Include appropriate controls (IgG, beads-only, single transfections)

• Proximity ligation assay (PLA) protocol:

  • Fix cells in 4% paraformaldehyde for 10 minutes

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% BSA

  • Incubate with PHOX2A-biotin antibody (1:400) and PHOX2B antibody (1:400)

  • Follow with appropriate PLA probes and detection reagents

  • This approach has successfully demonstrated PHOX2A-PHOX2B interactions in both nuclear and cytoplasmic compartments

• Transcriptional regulatory studies:

  • PHOX2A promoter-luciferase reporter assays with PHOX2B overexpression/knockdown

  • Dopamine β-hydroxylase promoter assays to study synergistic activation

  • ChIP assays to detect binding at endogenous regulatory regions

  • These approaches have shown that PHOX2B can activate the PHOX2A promoter, and both factors can synergistically activate target genes

• Analysis of mutant effects:

  • Compare wild-type PHOX2B with CCHS-associated mutations

  • Assess how mutations affect interaction with PHOX2A

  • Evaluate consequences for target gene expression

  • Research has shown different molecular mechanisms for NPARM and PARM mutations

What experimental approaches are recommended for studying PHOX2A in developmental contexts?

Developmental studies require specialized techniques to track PHOX2A expression across time:

• Lineage tracing strategies:

  • Combine PHOX2A immunostaining with neural crest lineage markers

  • In mouse models, use lineage-specific Cre drivers with reporter lines

  • Track temporal appearance of PHOX2A following PHOX2B expression during neuronal differentiation

• Ex vivo tissue analysis protocol:

  • For embryonic tissues, fix in 4% paraformaldehyde for 4-6 hours

  • Process for cryosectioning (optimal for preservation of epitopes)

  • Use the biotin-conjugated PHOX2A antibody at 1:100-250 dilution

  • Counterstain with developmental stage markers

• Differentiation model systems:

  • Neuroblastoma cell lines treated with retinoic acid provide accessible models

  • Neural crest-derived stem cells induced to differentiate

  • iPSC-derived neural crest and autonomic neurons

  • Monitor PHOX2A at both mRNA and protein levels across differentiation timeline

• Temporal considerations:

  • Include multiple timepoints to capture transient expression patterns

  • Be aware that PHOX2A protein may be degraded despite continued mRNA expression

  • Coordinate with developmental markers to standardize staging across experiments

Studies have demonstrated that PHOX2A expression follows PHOX2B during neuronal differentiation from neural crest, with cells that begin to differentiate along a neuronal lineage continuing to express PHOX2B and beginning to express PHOX2A .

How can I integrate PHOX2A antibody data with functional assays?

• Gene expression correlation analysis:

  • Pair PHOX2A immunostaining with qPCR for target genes

  • Analyze correlation between PHOX2A protein levels and expression of genes like tyrosine hydroxylase and dopamine-beta hydroxylase

  • Create correlation matrices across developmental timepoints or disease states

• Integrated workflow for neuroblastoma research:

  • Step 1: Characterize baseline PHOX2A/B expression by Western blot and qPCR

  • Step 2: Induce differentiation with retinoic acid and monitor changes over time

  • Step 3: Correlate molecular changes with morphological differentiation

  • Step 4: Assess functional outcomes (neurite outgrowth, electrophysiology)

  • This approach connects PHOX2A regulation with neuroblastoma differentiation status

• Respiratory function correlation in CCHS models:

  • Combine PHOX2A/B immunostaining of brainstem sections with whole-body plethysmography

  • Correlate PHOX2A expression in the retrotrapezoid nucleus with CO₂ sensitivity

  • In mouse models, relate PHOX2A expression patterns to breathing abnormalities

• Multi-omics integration strategy:

  • Layer immunohistochemistry data with transcriptomics and proteomics

  • Use computational approaches to identify PHOX2A-associated gene networks

  • Create visualization tools that connect molecular data with functional outcomes

Research on congenital central hypoventilation syndrome has demonstrated how PHOX2B mutations affect both molecular interactions with PHOX2A and functional outcomes in respiratory control, providing a model for integrating molecular and physiological data .