PHOX2A Antibody, HRP conjugated

What is PHOX2A and what is its biological significance?

PHOX2A (Paired-Like Homeobox 2A) is a transcription activator/factor that plays a crucial role in regulating the specificity of expression of catecholamine biosynthetic genes. This protein is integral to maintaining the noradrenergic phenotype in certain neuronal populations . It belongs to the paired homeobox family and is also known by several synonyms including ARIX, PMX2A, and Aristaless homeobox protein homolog . PHOX2A primarily functions within the nucleus, where it binds to specific DNA sequences to influence gene expression patterns critical for proper neuronal development and function .

What is the advantage of using HRP-conjugated PHOX2A antibodies compared to unconjugated versions?

HRP (Horseradish Peroxidase) conjugation provides direct detection capability without requiring secondary antibodies, which streamlines experimental workflows and potentially reduces background signal. HRP-conjugated PHOX2A antibodies enable one-step detection in applications like ELISA, Western blotting, and immunohistochemistry . This direct detection system is particularly valuable when working with limited samples or when experimental designs require multiple antibody labeling approaches. Additionally, HRP conjugation enables highly sensitive detection through enzymatic amplification of signal, which can be advantageous when working with low-abundance transcription factors like PHOX2A.

What epitope regions of PHOX2A are typically targeted by commercially available antibodies?

Multiple epitope regions are targeted by different PHOX2A antibodies, offering researchers flexibility in experimental design. Commercial antibodies target various amino acid sequences including:

The choice of epitope can significantly affect antibody performance in different applications, particularly for proteins that may have multiple isoforms or undergo post-translational modifications .

What are the validated applications for HRP-conjugated PHOX2A antibodies?

HRP-conjugated PHOX2A antibodies have been validated for several experimental applications, with specificity depending on the particular antibody:

Researchers should note that application validation varies between manufacturers, and preliminary testing is recommended when using these antibodies in novel experimental contexts. When selecting an antibody for a specific application, consider those with explicit validation data rather than predicted reactivity .

What are the recommended dilutions and protocols for Western blot applications?

While specific dilution recommendations for HRP-conjugated PHOX2A antibodies are not provided in all sources, related antibody clones provide useful guidance. For example, the ab155084 antibody (same clone as ab249179) has been successfully used at 1/1000 dilution for Western blotting with SH-SY-5Y and Neuro-2a cell lysates (10 μg loading), with detection using Goat anti-rabbit HRP secondary antibody at 1/2000 dilution .

For HRP-conjugated versions, researchers should typically begin with manufacturer recommendations and optimize from there, generally starting with more dilute concentrations for direct detection systems. Standard Western blotting protocols apply, including proper sample preparation, gel separation, transfer to membrane, blocking, primary antibody incubation, washing, and detection using appropriate substrates for HRP visualization .

How should researchers optimize PHOX2A antibody usage for immunofluorescence studies?

For immunofluorescence applications, researchers can reference protocols used with related PHOX2A antibodies. The ab155084 antibody has been successfully used for immunofluorescent analysis of SH-SY-5Y cells at a 1/100 dilution . When using HRP-conjugated antibodies for immunofluorescence, researchers should:

• Optimize fixation methods appropriate for nuclear transcription factors (typically 4% paraformaldehyde)

• Include permeabilization steps to ensure nuclear access (0.1-0.5% Triton X-100)

• Use appropriate blocking solutions to minimize background

• Utilize tyramide signal amplification systems for converting HRP activity to fluorescent signal

• Include appropriate controls, particularly nuclear counterstains like DAPI to confirm nuclear localization expected for PHOX2A

Cell lines with known PHOX2A expression, such as SH-SY-5Y and Neuro-2a, serve as excellent positive controls for optimization .

What controls should be included when using PHOX2A antibodies in experimental settings?

Robust experimental design when using PHOX2A antibodies should incorporate several controls:

• Positive controls: Cell lines with established PHOX2A expression such as SH-SY-5Y and Neuro-2a

• Negative controls: Cell lines known to lack PHOX2A expression or tissues from knockout models

• Isotype controls: Particularly important for immunohistochemistry applications to assess non-specific binding

• Peptide competition assays: To confirm epitope specificity of the antibody

• Secondary-only controls: For non-HRP conjugated versions, to establish background signal levels

• Multiple antibody validation: Using antibodies targeting different epitopes of PHOX2A to confirm specificity

For advanced studies, researchers may consider using transgenic systems like Phox2a::Cre mice crossed with reporter lines as used in recent deep sequencing studies .

What are common causes of non-specific binding and false positives when using PHOX2A antibodies?

Several factors can contribute to non-specific binding and false positives:

• Insufficient blocking: Optimize blocking conditions, typically using 1-5% BSA or serum from the species of the secondary antibody

• Improper antibody dilution: Over-concentrated antibody solutions increase background

• Cross-reactivity with related homeobox proteins: Select antibodies targeting unique epitopes of PHOX2A

• Sample over-fixation: Excessive fixation can create artifacts, particularly in immunohistochemistry

• Endogenous peroxidase activity: For HRP-conjugated antibodies, include appropriate peroxidase quenching steps

• Inappropriate storage conditions: Follow manufacturer recommendations to store at -20°C and avoid freeze-thaw cycles

To minimize these issues, researchers should carefully optimize each experimental parameter and include appropriate controls as described above.

How can PHOX2A antibodies be integrated with single-cell analysis technologies?

PHOX2A antibodies can enhance single-cell analysis studies through several approaches:

• Fluorescence-activated cell sorting (FACS): Use fluorescently-labeled PHOX2A antibodies (or secondary detection of non-conjugated versions) to isolate PHOX2A-expressing cells for downstream analysis

• Single-cell Western blotting: Detect PHOX2A in individual cells to correlate with other phenotypic markers

• Mass cytometry (CyTOF): Incorporate metal-tagged PHOX2A antibodies into comprehensive cellular profiling panels

• Spatial transcriptomics validation: Use PHOX2A immunostaining to validate spatial transcriptomic data

Recent studies have employed sophisticated approaches to analyze Phox2a-expressing cells, such as single-nucleus RNA sequencing of Phox2a-GFP nuclei isolated from transgenic mice where Phox2a::Cre mice were crossed with reporter lines . This approach allowed researchers to identify five distinct classes of Phox2a-expressing neurons with different molecular signatures.

What considerations are important when using PHOX2A antibodies in chromatin immunoprecipitation (ChIP) studies?

When designing ChIP experiments to study PHOX2A DNA binding:

• Epitope accessibility: Select antibodies targeting epitopes that remain accessible when PHOX2A is bound to DNA

• Cross-linking optimization: Adjust formaldehyde concentration and incubation time to effectively capture protein-DNA interactions without overfixing

• Sonication parameters: Optimize to generate DNA fragments of appropriate size (typically 200-500 bp)

• Antibody validation: Confirm the ability of the selected PHOX2A antibody to recognize the native conformation in nuclear extracts

• Positive control regions: Include known PHOX2A binding sites in validation experiments

• Negative control regions: Include genomic regions not expected to bind PHOX2A

• Input normalization: Essential for accurate quantification of enrichment

These considerations are particularly important given PHOX2A's role as a transcription factor involved in regulating catecholamine biosynthetic genes .

What is known about PHOX2A conservation across species and how does this impact antibody selection?

PHOX2A demonstrates considerable evolutionary conservation, affecting both research applications and antibody selection:

This conservation profile suggests structural similarities in PHOX2A across species, particularly in functional domains. When selecting antibodies for cross-species studies, researchers should:

• Choose antibodies targeting highly conserved epitopes

• Validate antibody performance in each species of interest

• Consider epitopes that may differ between species when species-specific detection is required

• Reference sequence homology between the immunogen used and the target species' PHOX2A sequence

Proper species validation is essential since manufacturer predictions of cross-reactivity may be based on sequence homology rather than experimental validation .

How can researchers validate PHOX2A antibodies for species not explicitly tested by manufacturers?

For species not explicitly validated by manufacturers, researchers should implement a systematic validation approach:

• In silico analysis: Compare the epitope sequence with the PHOX2A sequence in the target species to assess potential homology

• Western blot validation: Run samples from the species of interest alongside positive control samples from validated species

• Blocking peptide competition: Confirm signal specificity using peptides corresponding to the target epitope

• Knockout/knockdown controls: When available, use genetic models to confirm antibody specificity

• Multiple antibody approach: Use several antibodies targeting different epitopes and compare results

• Immunoprecipitation-mass spectrometry: For definitive validation, perform IP followed by MS to confirm target identity

This methodical approach is particularly important when working with less-studied model organisms where validated reagents may be limited.

What are the optimal storage and handling conditions for maintaining HRP-conjugated PHOX2A antibody activity?

To maintain optimal activity of HRP-conjugated PHOX2A antibodies, researchers should adhere to these guidelines:

• Storage temperature: Store at -20°C as recommended by manufacturers

• Buffer composition: Products are typically supplied in buffered solutions containing glycerol (often 50%) and stabilizers

• Aliquoting: Divide into small aliquots upon receipt to avoid repeated freeze-thaw cycles

• Freeze-thaw cycles: Minimize as these can reduce HRP enzymatic activity and antibody binding capacity

• Light exposure: Limit exposure to light, particularly during storage

• Contamination prevention: Use sterile techniques when handling antibody solutions

• Dilution practices: Dilute only the amount needed for immediate use

• Long-term storage: For bs-11577R-HRP, storage buffer contains "0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol"

Proper storage and handling significantly impact experimental reproducibility and sensitivity when working with these specialized reagents.

What methods can be used to quantify and compare PHOX2A expression levels across different experimental conditions?

Researchers can employ several quantitative approaches when analyzing PHOX2A expression:

• Western blot densitometry: Quantify band intensity normalized to loading controls

• Quantitative immunofluorescence: Measure nuclear signal intensity across cells/tissues

• Flow cytometry: For single-cell quantification of PHOX2A levels in cell populations

• ELISA: For quantification in cell/tissue lysates

• qPCR correlation: Compare protein levels detected by antibodies with mRNA expression

• Single-cell analysis: As demonstrated in recent research using Phox2a-GFP nuclei sequencing

For accurate comparisons across conditions, researchers should:

• Maintain consistent antibody concentrations

• Process all samples simultaneously when possible

• Include standard curves where appropriate

• Normalize to appropriate housekeeping genes/proteins

• Use multiple technical and biological replicates

• Consider statistical approaches appropriate for the data distribution

When analyzing PHOX2A in complex tissues, recent deep sequencing studies have revealed five distinct classes of Phox2a-expressing neurons, suggesting that expression analysis should account for cellular heterogeneity .

How are PHOX2A antibodies being used to investigate neurodevelopmental disorders?

PHOX2A antibodies are valuable tools in studying neurodevelopmental disorders, particularly those involving autonomic nervous system dysfunction:

• CFEOM2 (Congenital Fibrosis of Extraocular Muscles Type 2): Some PHOX2A antibodies are specifically referenced in relation to this disorder , which is associated with PHOX2A mutations affecting cranial nerve development

• Autonomic nervous system disorders: Given PHOX2A's role in noradrenergic neuron development

• Neuroblastoma research: Investigating PHOX2A's role in this neural crest-derived tumor

• Developmental pathway analysis: Studying PHOX2A in the context of neural crest development and migration

Research approaches typically combine:

• Immunohistochemistry to assess PHOX2A expression patterns in affected tissues

• Cellular models examining the impact of disease-associated mutations

• Transgenic animal models, such as Phox2a::Cre mice crossed with reporter lines

• Co-expression studies examining PHOX2A in relation to other developmentally relevant factors

By correlating PHOX2A expression with disease phenotypes, researchers gain insights into pathological mechanisms and potential therapeutic interventions.

What are the considerations for multiplexing PHOX2A antibodies with other neural markers?

When designing multiplexed detection systems incorporating PHOX2A antibodies:

• Antibody compatibility: Select primary antibodies raised in different host species to avoid cross-reactivity

• Spectral overlap: For fluorescent detection, choose fluorophores with minimal spectral overlap

• Sequential detection: Consider sequential rather than simultaneous staining for challenging combinations

• HRP substrate selection: When using multiple HRP-conjugated antibodies, select substrates producing different colored precipitates

• Epitope retrieval optimization: Ensure retrieval conditions are compatible for all target epitopes

• Signal amplification balance: Adjust amplification methods to achieve comparable signal intensities

• Controls: Include single-stain controls to confirm specificity in the multiplexed context

Recent research demonstrates the value of combining PHOX2A detection with other markers, revealing that "Phox2a-GFP nuclei expressed high levels of the neuronal marker Meg3 and the vesicular glutamate transporter Slc17a6" with "little to no expression of the inhibitory neuronal markers Slc32a1 and Gad1" . This approach enabled researchers to characterize Phox2a-expressing neurons as primarily excitatory.

How should researchers interpret heterogeneous PHOX2A expression patterns in complex neural tissues?

Interpreting PHOX2A expression patterns requires consideration of several factors:

• Cellular heterogeneity: Recent single-nucleus RNA sequencing identified five distinct classes of Phox2a-expressing neurons , indicating that PHOX2A is not uniformly expressed across all neural populations

• Co-expression patterns: PHOX2A expression should be interpreted alongside other markers such as "Lypd1, Tacr1, and Tac1" that help define neuronal subclasses

• Developmental timing: Consider the developmental stage being examined, as PHOX2A expression may change temporally

• Spatial distribution: Analyze regional variations in expression patterns across different anatomical structures

• Quantitative assessment: Use appropriate image analysis tools to quantify nuclear PHOX2A signal intensity variations

• Single-cell resolution: When possible, analyze at single-cell level rather than tissue-level averages

Researchers should be particularly mindful that PHOX2A-expressing neurons appear to be primarily excitatory, as evidenced by high expression of vesicular glutamate transporter Slc17a6 and low expression of inhibitory markers Slc32a1 and Gad1 .

What statistical approaches are most appropriate for analyzing PHOX2A expression data across experimental groups?

When analyzing PHOX2A expression data:

• Distribution assessment: Test for normality to determine appropriate statistical tests

• Parametric vs. non-parametric: Choose tests based on data distribution characteristics

• Multiple comparisons: Apply appropriate corrections when comparing multiple groups

• Nested designs: Account for hierarchical data structures (e.g., cells within tissues within subjects)

• Correlation analyses: When examining relationships between PHOX2A and other markers

• Classification approaches: For identifying distinct cell populations based on PHOX2A and other markers

• Dimensionality reduction: Methods like PCA or t-SNE for visualizing complex multi-marker datasets

Advanced studies may employ computational approaches similar to those used in recent PHOX2A research, where "single-nucleus RNA sequencing and libraries were prepared using Smart-seq2" with subsequent analysis conducted "using Kallisto and further analysis was conducted using Seurat in R" .