PAX9 Antibody, Biotin conjugated

What is PAX9 and why is it a significant research target?

PAX9 is a transcription factor required for normal development of various structures including the thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of skull and larynx, as well as distal limbs . It contains a paired DNA-binding domain that interacts with specific DNA sequences to regulate gene expression. PAX9 is highly conserved during evolution, indicating its fundamental biological importance . Research on PAX9 is critical for understanding both developmental biology and pathological conditions, particularly tooth agenesis and certain cancers .

What applications is PAX9 Antibody, Biotin conjugated suitable for?

PAX9 antibodies with biotin conjugation are primarily used in immunological applications including ELISA, as specifically mentioned in product descriptions . While certain PAX9 antibodies have been validated for other applications such as immunocytochemistry/immunofluorescence (ICC/IF) and Western blotting (WB), the specific applications for biotin-conjugated variants may vary by manufacturer . The biotin conjugation provides enhanced detection sensitivity when used with avidin/streptavidin systems, making these antibodies particularly valuable for detecting low-abundance PAX9 in complex samples .

How does PAX9 antibody detection differ across tissue types?

PAX9 expression shows distinct patterns across different tissues. In control tissues, moderate to numerous (++/+++) PAX9-positive cells are typically observed in the epithelium, while connective tissues show minimal expression (0 to 0/+) . Significant differences have been documented in PAX9 expression between normal tissues and pathological samples such as cleft-affected tissues, with statistically significant variations (p < 0.001) observed between control groups and various cleft types . These expression patterns provide important biological context for researchers investigating developmental abnormalities.

What controls should be included when using PAX9 Antibody, Biotin conjugated?

When designing experiments with biotin-conjugated PAX9 antibodies, multiple controls are essential for result validation:

• Positive controls: Include samples known to express PAX9, such as developing tooth tissues or certain cancer cell lines (e.g., SCLC lines described in the literature)

• Negative controls: Use tissues or cells where PAX9 has been knocked down via RNAi (similar to the approaches mentioned for PAX9 validation in cancer studies)

• Isotype controls: Include matched isotype antibodies (such as rabbit IgG for polyclonal rabbit antibodies) conjugated with biotin

• Endogenous biotin blocking: Apply avidin/biotin blocking steps in biotin-rich tissues

• Secondary-only controls: Omit primary antibody to assess non-specific binding of detection systems

Incorporating these controls ensures reliable interpretation of PAX9 immunodetection results.

How should sample preparation be optimized for PAX9 antibody detection?

Optimal sample preparation is crucial for successful PAX9 detection. Recommendations include:

• Fixation: Use 10% neutral buffered formalin for paraffin-embedded sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended for most PAX9 antibodies

• Blocking: Block endogenous biotin with avidin/biotin blocking kit when using biotin-conjugated antibodies

• Dilution optimization: Determine optimal antibody concentration through titration experiments (manufacturer recommendations suggest end-user optimization)

• Incubation conditions: Typically overnight incubation at 4°C yields best results for nuclear transcription factors like PAX9

These preparation steps significantly impact staining specificity and sensitivity.

What factors affect the stability and performance of PAX9 Antibody, Biotin conjugated?

Several factors influence antibody stability and performance:

• Storage conditions: Aliquot and store at -20°C; avoid repeated freeze/thaw cycles and exposure to light

• Buffer composition: Typically supplied in PBS pH 7.4 with stabilizers (0.03% Proclin-300) and 50% glycerol

• Shelf-life: Generally stable for 12 months when properly stored

• Sample type compatibility: Confirm compatibility with your specific sample type (fixed vs. frozen, human vs. mouse, etc.)

• Detection system: Use freshly prepared streptavidin-conjugated detection reagents

Adherence to these guidelines ensures optimal antibody performance in experimental applications.

How can PAX9 Antibody, Biotin conjugated be used to investigate PAX9 mutations associated with tooth agenesis?

PAX9 mutations are significantly linked to tooth agenesis. Biotin-conjugated PAX9 antibodies can be valuable tools for investigating these mutations through:

• Comparative expression analysis: Detecting differential expression between wild-type and mutant PAX9 in patient-derived samples

• Domain-specific detection: Utilizing antibodies targeting specific regions to distinguish between different mutant forms (nine novel mutations have been identified in the paired DNA-binding domain)

• Functional studies: Combining with reporter assays to correlate protein detection with transcriptional activity impairment

• Co-localization studies: Investigating altered nuclear localization of mutant PAX9 proteins

Research has shown that different PAX9 mutations (missense, nonsense, frameshift) have varying effects on protein expression and function, with some mutations rendering the protein undetectable while others produce detectable but non-functional proteins .

What methodological approaches can be used to study PAX9's role in cancer progression?

PAX9 has been identified as an essential factor in small cell lung cancer (SCLC), where it is overexpressed in malignant tumor samples . To investigate PAX9's oncogenic functions, researchers can employ biotin-conjugated PAX9 antibodies in:

• Chromatin immunoprecipitation (ChIP): To identify genomic regions where PAX9 binds to enhancer elements

• Co-immunoprecipitation: To confirm PAX9 interaction with the nucleosome remodeling and deacetylase (NuRD) complex

• Immunohistochemistry: To evaluate PAX9 expression across tumor stages and correlate with clinical outcomes

• Proximity ligation assays: To visualize PAX9 interactions with epigenetic regulators in situ

These approaches can help elucidate the mechanisms by which PAX9 represses enhancer activity and affects tumor-suppressive gene expression .

How can researchers investigate the epigenetic regulation mechanisms of PAX9?

PAX9 functions within complex epigenetic regulatory networks. Biotin-conjugated PAX9 antibodies can facilitate studies of these mechanisms through:

• Sequential ChIP assays: To map co-occupancy of PAX9 with histone modifications at enhancer elements

• Mass spectrometry following immunoprecipitation: To identify novel PAX9 interacting partners

• CUT&RUN or CUT&Tag methods: For high-resolution mapping of PAX9 binding sites

• Combinatorial studies with HDAC inhibitors: To assess reversal of PAX9-mediated gene repression

Research has demonstrated that PAX9 interacts with the NuRD complex at enhancers to repress nearby gene expression, which can be reversed by pharmacologic HDAC inhibition . This suggests potential therapeutic approaches targeting PAX9-regulated epigenetic mechanisms.

How should researchers quantify and standardize PAX9 immunostaining results?

For reproducible and comparable results, PAX9 immunostaining should be quantified using standardized methods:

• Scoring systems: Use established semiquantitative scoring (0 to +++) as demonstrated in published research on PAX9 expression (e.g., no positive cells (0), barely detectable (+), moderate (++), or numerous (+++) positive structures)

• Digital image analysis: Employ software-based quantification for objective assessment of staining intensity and percentage of positive cells

• Internal reference standards: Include calibration samples in each experiment to normalize between batches

• Blinded evaluation: Have multiple observers score samples independently to reduce bias

• Statistical analysis: Apply appropriate statistical tests (e.g., Kruskal-Wallis H test and Mann-Whitney U test for non-parametric data) to determine significance of differences

These approaches ensure consistent interpretation across different experimental conditions and laboratories.

What are common troubleshooting strategies for weak or nonspecific PAX9 antibody signals?

When encountering issues with PAX9 immunodetection, consider these troubleshooting approaches:

• For weak signals:

  • Optimize antigen retrieval methods (test multiple buffers and durations)

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Verify sample integrity and PAX9 expression levels

• For nonspecific signals:

  • Increase blocking duration and concentration

  • Optimize antibody dilution

  • Reduce incubation temperature

  • Include additional washing steps

  • Pre-absorb antibody with recombinant PAX9 protein to confirm specificity

• For inconsistent results:

  • Standardize tissue processing and fixation protocols

  • Verify antibody lot consistency

  • Control for endogenous biotin

  • Check detection reagent functionality and freshness

These strategies address the most common technical challenges in PAX9 immunodetection experiments.

How can researchers validate that their PAX9 antibody recognizes the intended target?

Validation of antibody specificity is critical for reliable research outcomes. Methods include:

• Genetic approaches: Test antibody in PAX9 knockout/knockdown models (such as those generated using shRNA constructs targeting PAX9)

• Western blot analysis: Confirm single band of expected molecular weight (paired box protein Pax-9 is approximately 36-38 kDa)

• Peptide competition assays: Pre-incubate antibody with recombinant PAX9 protein to confirm specific binding

• Multiple antibody comparison: Compare staining patterns using different PAX9 antibodies targeting distinct epitopes

• RNA-protein correlation: Compare protein detection with mRNA expression data from real-time PCR using primers specific for PAX9

These validation approaches ensure that experimental findings accurately reflect PAX9 biology rather than antibody artifacts.

How does PAX9 expression correlate with craniofacial development abnormalities?

PAX9 plays a crucial role in craniofacial development, and its expression patterns provide insights into developmental abnormalities:

• Statistical analyses have demonstrated significant differences in PAX9-positive structures between control tissues and various types of cleft-affected tissues (unilateral cleft lip, bilateral cleft lip, and isolated cleft palate) with p < 0.001

• The observed differential expression patterns between epithelium and connective tissue suggest tissue-specific regulatory roles during development

• PAX9 expression patterns can be correlated with specific developmental anomalies to establish genotype-phenotype relationships

• Mutations in the PAX9 gene have been directly linked to nonsyndromic tooth agenesis, demonstrating its critical role in dental development

These correlations help establish PAX9 as an important factor in the etiology of craniofacial developmental disorders.

What is the relationship between PAX9 expression and transcriptional regulation in cancer progression?

PAX9 functions as a transcriptional regulator with significant implications in cancer:

• Genome-wide studies have revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity

• In small cell lung cancer, genetic depletion of PAX9 leads to significant induction of a primed-active enhancer transition, resulting in increased expression of neural differentiation and tumor-suppressive genes

• The BAP1/ASXL3/BRD4 epigenetic axis drives PAX9 transcription in SCLC, creating a regulatory network that supports cancer progression

• Mechanistically, PAX9 interacts with the NuRD complex at enhancers to repress nearby gene expression, creating a targetable epigenetic vulnerability

These findings highlight PAX9's role as an oncogenic factor and suggest potential therapeutic approaches targeting PAX9-regulated networks.

How can PAX9 study contribute to understanding evolutionary conservation of developmental processes?

PAX9 research provides valuable insights into evolutionary conservation:

• Conservation analysis has revealed that key amino acids in the PAX9 paired DNA-binding domain (including Arg26, Arg47, Ile56, and Ala108) are highly conserved during evolution, indicating their fundamental importance to protein function

• Mutations in these conserved regions consistently disrupt DNA binding ability and transcriptional activation of target genes, demonstrating functional conservation

• Comparative studies across species using PAX9 antibodies can reveal evolutionary adaptations in developmental regulatory networks

• The high degree of conservation suggests that findings from animal models may have translational relevance to human developmental disorders

Understanding this evolutionary conservation helps establish fundamental principles of developmental biology that transcend species boundaries.

Table 1: PAX9 Mutation Types and Their Functional Effects

Data compiled from mutation analysis studies of PAX9 in tooth agenesis patients

Table 2: PAX9 Antibody, Biotin Conjugated Properties

Data derived from product specifications for commercially available PAX9 antibodies