PAX9 Antibody, HRP conjugated

What is PAX9 and why is it an important research target?

PAX9 is a sequence-specific DNA binding transcription factor belonging to the paired box (PAX) family. It contains a paired box domain, a paired type homeodomain, and an octapeptide sequence . PAX9 functions as an RNA Polymerase II transcription factor that regulates the expression of proteins required for craniofacial and tooth development in humans . It is essential for normal development of the thymus, parathyroid glands, teeth, portions of the skull, larynx, and distal limbs .

Recent research has revealed that PAX9 may function as a tumor suppressor gene in cervical cancer by modulating cell proliferation and apoptosis . Additionally, a genome-wide screen in small cell lung cancer has shown PAX9's involvement in epigenetic enhancer silencing and tumor progression through interaction with the nucleosome remodeling and deacetylase (NuRD) complex .

What applications are PAX9 antibodies typically used for in research?

PAX9 antibodies are employed in multiple experimental applications:

• Western Blotting (WB): For detecting PAX9 protein expression levels in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing PAX9 localization in paraffin-embedded (IHC-P) or frozen (IHC-F) tissue sections

• Immunofluorescence (IF): For cellular localization studies

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of PAX9

• Immunoprecipitation (IP): For isolating PAX9 protein complexes

• Immunocytochemistry (ICC): For detecting PAX9 in cultured cells

What is the advantage of using HRP-conjugated PAX9 antibodies compared to unconjugated versions?

HRP-conjugated PAX9 antibodies offer several methodological advantages:

• Direct detection without the need for secondary antibodies, reducing potential cross-reactivity and background signal

• Simplified experimental workflow with fewer incubation and washing steps

• Enhanced sensitivity when used with chemiluminescent substrates

• Reduced experiment time by eliminating secondary antibody incubation

• Greater consistency in signal generation across experiments

In Western blot applications, PAX9 antibody-HRP conjugates allow for direct visualization of the target protein (approximately 36 kDa) without requiring additional detection reagents .

What are the optimal dilution ratios for PAX9 antibody-HRP conjugates in different experimental applications?

Based on the technical specifications from multiple antibody vendors, the recommended dilution ratios vary by application:

Most vendors recommend that optimal dilutions should be determined by the end user based on specific experimental conditions .

How should I properly store and handle PAX9 antibody-HRP conjugates to maintain reactivity?

To maintain optimal activity of PAX9 antibody-HRP conjugates:

• Store at -20°C in small aliquots to avoid repeated freeze/thaw cycles

• When working with the antibody, keep it on ice

• Protect from prolonged exposure to light, which can affect the HRP moiety

• Store in glycerol-containing buffers (typically 50% glycerol) to prevent freeze damage

• For long-term storage beyond 12 months, maintain at -80°C

• Always centrifuge briefly before opening the vial to collect liquid at the bottom

Most manufacturers guarantee the stability of these antibodies for 12 months from the date of receipt when stored properly .

What controls should be included when using PAX9 antibody-HRP conjugates in experimental designs?

A robust experimental design with PAX9 antibody-HRP conjugates should include:

• Positive control: Tissues or cell lines known to express PAX9 (e.g., MCF10A cells, rat heart tissue)

• Negative control: Either PAX9-null tissues/cells or isotype control antibodies

• Peptide competition assay: Pre-incubation with the immunizing peptide should abolish specific staining

• Loading control: For Western blot applications, include detection of housekeeping proteins

• Knockdown validation: If possible, include samples with PAX9 knockdown using shRNA or CRISPR-Cas9 to verify antibody specificity

Research indicates that PAX9 expression can be detected in human breast cancer tissues, making these suitable positive controls for antibody validation .

How can I distinguish between true PAX9 signal and cross-reactivity with other PAX family members when using PAX9 antibody-HRP conjugates?

PAX family proteins share significant homology, particularly in their paired box domains, making specific detection challenging. To ensure specificity:

• Select antibodies raised against regions with lowest homology to other PAX family members

• Use antibodies validated through knockout/knockdown experiments

• Employ epitope-specific antibodies targeting unique regions of PAX9

• Perform parallel experiments with antibodies recognizing different epitopes of PAX9

• Include PAX family member-specific controls in your experiments

For example, CentriMo enrichment analysis of sequences upstream of PAX9-regulated genes reveals significant enrichment of binding sequences for PAX3, PAX5, PAX6, and PAX7 DNA binding domains. Multiple PAX proteins can bind the same DNA sequence, making verification of specificity critical .

What are the challenges in detecting nuclear localization of PAX9 protein and how can I optimize protocols to overcome them?

PAX9 functions primarily as a nuclear transcription factor, but detecting its nuclear localization can be challenging due to:

• Fixation artifacts: Over-fixation can mask epitopes through protein cross-linking

• Nuclear membrane permeabilization: Insufficient permeabilization prevents antibody access

• Low expression levels: PAX9 may be expressed at levels below detection threshold

• Variant-specific localization differences: Some PAX9 variants show altered subcellular localization

Optimization strategies:

• Use antigen retrieval methods such as microwave treatment with 10 mM Tris/EDTA buffer pH 9.0 before IHC staining

• Employ fluorescent secondary detection systems for enhanced sensitivity

• Include nuclear counterstains to clearly demarcate nuclear boundaries

• Utilize confocal microscopy for higher resolution imaging

Recent research has shown that certain PAX9 variants (Ser49Leu) show reduced nuclear localization compared to wild-type PAX9, which may affect experimental outcomes .

How can I verify if my PAX9 antibody-HRP conjugate is detecting functional versus non-functional PAX9 protein variants?

To distinguish between functional and non-functional PAX9 variants:

• Complementary functional assays: Pair antibody detection with reporter gene assays measuring PAX9 transcriptional activity

• Co-immunoprecipitation: Verify interactions with known PAX9 binding partners (e.g., NuRD complex)

• ChIP-seq analysis: Confirm DNA binding capacity of detected PAX9 protein

• Domain-specific antibodies: Use antibodies targeting specific functional domains

• Mutation analysis: Compare detection in samples with known PAX9 mutations

Recent studies have shown that PAX9 interacts with the NuRD complex at enhancers to repress nearby gene expression. Mutations affecting this interaction could produce non-functional protein variants that still react with antibodies .

How can PAX9 antibody-HRP conjugates be used to investigate PAX9's role in cancer progression and tumor suppression?

PAX9 has demonstrated tumor suppressor activity in cervical cancer, making it an important research target. Methods to investigate this role include:

• Expression correlation studies: Using PAX9 antibody-HRP conjugates in IHC to correlate PAX9 expression with clinical outcomes in tumor tissue microarrays

• Molecular mechanism investigation: Western blot analysis to monitor changes in apoptosis-related proteins (Bcl-2, Bax, cleaved-caspase-3) following PAX9 manipulation

• In vivo tumor models: IHC staining of xenograft tumors to assess PAX9 expression and its correlation with tumor progression

• Cell proliferation markers: Dual staining with PAX9 and Ki-67 to examine the relationship between PAX9 expression and proliferative capacity

In cervical cancer research, PAX9 was found to inhibit proliferation of cancer cell lines and promote apoptosis, thereby suppressing tumor growth in vivo. This was associated with the up-regulation of cleaved-caspase-3 and Bax and the down-regulation of Bcl-2 .

What methodological approaches can be used to study the interaction between PAX9 and the NuRD complex using PAX9 antibody-HRP conjugates?

The interaction between PAX9 and the NuRD complex represents an important epigenetic regulatory mechanism. To study this interaction:

• Co-immunoprecipitation (Co-IP): Use PAX9 antibodies to pull down protein complexes, followed by Western blot with HRP-conjugated PAX9 antibodies and NuRD complex component antibodies

• Chromatin immunoprecipitation (ChIP): Employ PAX9 antibodies to identify genomic binding sites, then correlate with NuRD complex occupancy

• Proximity ligation assay (PLA): Visualize in situ protein-protein interactions between PAX9 and NuRD components

• HDAC inhibition studies: Examine how pharmacological HDAC inhibition affects PAX9-mediated repression

Research has shown that PAX9 interacts with the NuRD complex at enhancers to repress nearby gene expression, which can be reversed by pharmacologic HDAC inhibition. This interaction provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human small cell lung cancer .

How can PAX9 antibody-HRP conjugates be used to investigate developmental anomalies associated with PAX9 mutations?

PAX9 plays crucial roles in embryonic development, and mutations can lead to developmental disorders:

• Genotype-phenotype correlation: Use IHC with PAX9 antibody-HRP conjugates to examine expression patterns in tissue samples from individuals with PAX9 mutations

• Animal models: Study expression patterns in PAX9 mutant mouse models with skeletal and dental abnormalities

• Protein localization studies: Investigate how mutations affect nuclear localization of PAX9 protein

• Molecular pathway analysis: Examine downstream effects on Wnt signaling and other developmental pathways

Recent genotype-phenotype pattern analysis of pathogenic PAX9 variants revealed that mutations affecting the paired box domain can significantly impact protein function and cause developmental anomalies. Immunofluorescence assays showed that certain PAX9 variants (e.g., Ser49Leu) have reduced nuclear localization compared to wild-type PAX9, which may contribute to their pathogenicity .

What are the technical considerations for using PAX9 antibody-HRP conjugates in multiplex immunoassays?

Multiplex detection involving PAX9 requires careful technical planning:

• Antibody compatibility: Ensure primary antibodies are from different host species to prevent cross-reactivity

• Signal separation: When using multiple HRP-conjugated antibodies, employ sequential detection with HRP inactivation between steps

• Blocking optimization: Use protocols that minimize background without affecting epitope recognition

• Counterstain selection: Choose counterstains that won't interfere with HRP detection systems

• Control panels: Include appropriate single-stain controls to verify specificity in multiplex settings

For example, when studying PAX9's role in cancer, researchers might want to simultaneously detect PAX9 and proliferation markers. This requires careful antibody selection and optimization of staining protocols to minimize cross-reactivity while maximizing specific signal detection .

What are common causes of non-specific background when using PAX9 antibody-HRP conjugates and how can they be mitigated?

Non-specific background is a common challenge with HRP-conjugated antibodies. Causes and solutions include:

For PAX9 detection specifically, background issues can be reduced by using antibodies raised against unique regions of PAX9 that have less homology with other PAX family members .

How can I determine if inconsistent results with PAX9 antibody-HRP conjugates are due to technical issues or biological variability?

To distinguish between technical and biological sources of variability:

• Technical controls: Include the same positive control sample in each experiment

• Standardization: Normalize signal to loading controls or housekeeping proteins

• Batch testing: When possible, process all experimental samples in the same batch

• Antibody validation: Periodically verify antibody performance using known standards

• Biological replicates: Use sufficient biological replicates to account for natural variation

• Technical replicates: Perform multiple technical replicates to assess methodology precision

For PAX9 specifically, expression can vary significantly between tissue types and developmental stages. Validation studies showed strong PAX9 expression in rat heart tissue and various cell lines, which can serve as reliable positive controls .

Why might I detect multiple bands or unexpected molecular weight bands when using PAX9 antibody-HRP conjugates in Western blotting?

Detection of unexpected bands may have several explanations:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter apparent molecular weight

• Alternative splicing: PAX9 may have multiple isoforms with different molecular weights

• Proteolytic degradation: Sample preparation issues may lead to protein degradation products

• Non-specific binding: Cross-reactivity with related proteins, especially other PAX family members

• Antibody degradation: Storage conditions may affect antibody specificity

The expected molecular weight of PAX9 is approximately 36-38 kDa . When encountering unexpected bands, verification experiments such as knockdown/knockout controls, competition assays with immunizing peptides, or comparison with alternative PAX9 antibodies should be conducted .

How can PAX9 antibody-HRP conjugates be employed in studying epigenetic regulation mechanisms involved in PAX9 function?

PAX9's role in epigenetic regulation can be investigated through:

• ChIP-seq analysis: Use PAX9 antibodies to identify genomic regions bound by PAX9, followed by analysis of associated histone modifications

• Sequential ChIP: Perform ChIP with PAX9 antibodies followed by ChIP with antibodies against histone modifications or other chromatin regulators

• Epigenetic inhibitor studies: Examine how PAX9 binding is affected by treatments with HDAC inhibitors or other epigenetic modulators

• Enhancer activation assessment: Correlate PAX9 binding with enhancer activation states using H3K27ac or other enhancer-associated marks

Recent research has shown that PAX9 interacts with the NuRD complex at enhancers to repress nearby gene expression. PAX9 depletion led to significant induction of a primed-active enhancer transition, resulting in increased expression of neural differentiation and tumor-suppressive genes .

What considerations should be made when designing experiments to study PAX9's role in ribosome biogenesis using PAX9 antibody-HRP conjugates?

PAX9 has recently been implicated in ribosome biogenesis, requiring specific experimental approaches:

• Nucleolar localization studies: Use immunofluorescence to examine PAX9 localization relative to nucleolar markers

• Pre-rRNA processing analysis: Pair PAX9 detection with Northern blotting to examine effects on pre-rRNA processing intermediates

• PAX9 knockdown effects: Analyze how PAX9 depletion affects nucleolar number and morphology

• Downstream target assessment: Examine how PAX9 manipulation affects expression of nucleolar proteins

Research has shown that PAX9 depletion results in significant changes in pre-rRNA processing, including an increase in the 30S pre-rRNA intermediate and a decrease in the levels of its 21S processing product. Furthermore, 17.7% of mRNAs differentially expressed following PAX9 depletion code for proteins designated as nucleolar, with 62.4% of these being downregulated upon PAX9 depletion .

How can advances in microscopy techniques enhance the application of PAX9 antibody-HRP conjugates in developmental biology research?

Advanced microscopy techniques offer new possibilities for PAX9 research:

• Super-resolution microscopy: Provides nanometer-scale resolution of PAX9 localization within nuclear subcompartments

• Live-cell imaging: When combined with genetically encoded tags, allows for real-time tracking of PAX9 dynamics

• Tissue clearing techniques: Enables whole-mount imaging of PAX9 expression patterns in 3D

• Correlative light and electron microscopy (CLEM): Combines the specificity of immunolabeling with ultrastructural context

• Quantitative image analysis: Allows precise measurement of PAX9 expression levels across different cell types and developmental stages

For developmental studies, these techniques can help map PAX9 expression patterns in relation to morphological features during critical developmental windows, providing insight into how PAX9 mutations lead to developmental anomalies such as oligodontia and skeletal disorders .