PAX9 Antibody, FITC conjugated

What is PAX9 and what biological processes is it involved in?

PAX9 is a transcription factor that plays crucial roles in embryonic development and cellular differentiation. It is required for normal development of the thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of the skull and larynx, as well as distal limbs . As a member of the paired box (PAX) family of transcription factors, PAX9 contains a DNA-binding domain that recognizes specific sequences to regulate gene expression. In recent research, PAX9 has been implicated in cancer progression through its interaction with the nucleosome remodeling and deacetylase (NuRD) complex, where it functions at enhancers to repress nearby gene expression . This epigenetic regulation function makes PAX9 particularly interesting in developmental biology and cancer research contexts.

What specific applications are most suitable for PAX9 antibody with FITC conjugation?

The FITC-conjugated PAX9 antibody is particularly well-suited for applications requiring direct fluorescent detection without secondary antibodies. Based on technical specifications, this antibody has been validated for immunohistochemistry (IHC) and Western blotting (WB) . The FITC conjugation (Fluorescein Isothiocyanate) makes it especially valuable for immunofluorescence microscopy, flow cytometry, and confocal imaging applications. The direct fluorescence detection eliminates potential cross-reactivity issues associated with secondary antibodies and enables cleaner multi-color immunostaining protocols. When using this antibody for fluorescence applications, researchers should implement proper controls to account for tissue autofluorescence, which can overlap with the FITC emission spectrum.

What species reactivity should researchers expect with the N-terminal PAX9 antibody?

Based on immunogen sequence homology analysis, the N-terminal region PAX9 antibody is predicted to react with multiple species including Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse, Rabbit, and Zebrafish . The high cross-reactivity is due to the conserved nature of the N-terminal region of PAX9 across species. Specifically, the antibody's immunogen is a synthetic peptide with the sequence: LPGAIGGSKPRVTTPTVVKHIRTYKQRDPGIFAWEIRDRLLADGVCDKYN . Sequence alignment data indicates 100% homology for most mammalian species and 92% for zebrafish . This broad species reactivity makes the antibody versatile for comparative studies across model organisms, though researchers should validate reactivity in their specific experimental system before conducting full-scale experiments.

How should researchers optimize storage conditions for PAX9 antibody (FITC conjugated) to maintain its functionality?

For optimal preservation of FITC-conjugated PAX9 antibody functionality, researchers must store the antibody in light-protected vials or cover them with a light-protecting material such as aluminum foil to prevent photobleaching of the FITC fluorophore . The conjugated antibody remains stable for at least 12 months when stored at 4°C. For extended storage periods (up to 24 months), the antibody should be diluted with up to 50% glycerol and stored at -20°C to -80°C . It's important to note that repeated freezing and thawing cycles will compromise both enzyme activity and antibody binding capacity. Therefore, researchers should consider aliquoting the antibody before storage to minimize freeze-thaw cycles. Additionally, when working with the antibody, exposure to room light should be minimized during experimental procedures to prevent fluorophore degradation.

What controls are essential when using PAX9 antibody (FITC conjugated) in immunofluorescence studies?

When conducting immunofluorescence studies with FITC-conjugated PAX9 antibody, researchers should implement the following controls:

• Positive control: Use cell lines or tissues with confirmed PAX9 expression such as Jurkat cells, which are supported by BioGPS gene expression data to express PAX9 .

• Negative control: Include samples where PAX9 is known to be absent or use skeletal muscle tissue which shows minimal background staining compared to esophageal tissue (a PAX9-expressing tissue) .

• Isotype control: Include a FITC-conjugated rabbit IgG (for polyclonal antibodies) with the same concentration as the PAX9 antibody to assess non-specific binding.

• Blocking peptide control: Use the specific blocking peptide (Catalog # AAP34270) to confirm antibody specificity .

• Autofluorescence control: Include an unstained sample to assess natural tissue autofluorescence in the FITC channel.

• Subcellular localization verification: PAX9 should demonstrate nuclear localization consistent with its function as a transcription factor, as evident in immunofluorescence studies showing strong nuclear staining .

These controls help distinguish specific PAX9 signal from background and validate experimental findings, especially important when studying PAX9 mutations or expression changes in different biological contexts.

What dilution and detection parameters should be used for optimal results with PAX9 antibody (FITC conjugated)?

For optimal detection using PAX9 antibody (FITC conjugated), the following parameters are recommended based on technical specifications and research applications:

When analyzing the results, researchers should be aware that PAX9 exhibits nuclear localization as demonstrated in confocal imaging studies . For optimal visualization of nuclear staining, counterstaining with DAPI or another nuclear stain that doesn't overlap with FITC emission spectrum is recommended. Additionally, photobleaching should be minimized during image acquisition by using appropriate anti-fade mounting media and optimized exposure settings.

How can researchers utilize PAX9 antibody to investigate gene mutations and their functional consequences?

Researchers investigating PAX9 mutations can employ the FITC-conjugated PAX9 antibody in multiple sophisticated approaches:

• Mutation-specific protein detection: Using site-directed mutagenesis to generate PAX9 mutants (such as R26W, R47P, I56N, and A108P) followed by transfection into relevant cell lines, researchers can employ immunofluorescence microscopy with the PAX9 antibody to assess changes in protein localization, stability, or expression levels of these mutants compared to wild-type PAX9 .

• Functional domain analysis: The N-terminal region targeted by the antibody is critical for PAX9 function. Researchers can use the antibody to study how mutations in different domains affect protein-protein interactions, particularly with the nucleosome remodeling and deacetylase (NuRD) complex, which PAX9 interacts with to regulate enhancer activity .

• Comparative analysis with mRNA expression: By combining immunofluorescence or Western blot detection using the PAX9 antibody with real-time PCR analysis of PAX9 mRNA (using primers such as PAX9-F: 5′-AACCAGCTGGGAGGAGTGTT-3′ and PAX9-R: 5′-TGATGTCACACGGTCGGATG-3′), researchers can investigate post-transcriptional regulation of mutant PAX9 proteins .

• DNA binding capacity assessment: The PAX9 antibody can be used in chromatin immunoprecipitation (ChIP) assays to compare the DNA binding capacity of wild-type versus mutant PAX9 proteins, complementing gel shift analysis findings that show certain mutations (R26W, R47P, I56N, A108P, and 592delG) lead to loss of DNA binding ability .

This multi-faceted approach allows researchers to comprehensively characterize how specific mutations impact PAX9 protein function, stability, and downstream pathways.

What techniques can be combined with PAX9 immunodetection to investigate epigenetic regulation mechanisms?

To investigate PAX9's role in epigenetic regulation, researchers can employ several advanced techniques in combination with PAX9 immunodetection:

• ChIP-seq with PAX9 antibody: This approach can identify genome-wide PAX9 binding sites, particularly at enhancer regions where PAX9 has been shown to function with the NuRD complex to repress gene expression .

• Sequential ChIP (Re-ChIP): Using PAX9 antibody followed by antibodies against NuRD complex components can confirm co-occupancy at specific genomic loci.

• Co-immunoprecipitation (Co-IP): PAX9 antibody can be used in IP experiments to pull down PAX9 and associated proteins, followed by Western blot analysis to detect NuRD complex components and other potential interaction partners .

• HDAC inhibitor studies: Since PAX9 functions with the NuRD complex (which contains HDAC activity), researchers can perform immunofluorescence studies with PAX9 antibody in cells treated with HDAC inhibitors to observe changes in target gene expression or chromatin accessibility .

• CUT&RUN or CUT&Tag: These techniques offer higher resolution than ChIP-seq and can be performed with the PAX9 antibody to map PAX9 binding sites with greater precision and lower background.

• Proximity ligation assay (PLA): This technique can visualize and quantify interactions between PAX9 and NuRD complex components or other chromatin regulators in situ.

These combined approaches can provide mechanistic insights into how PAX9 contributes to "primed-active enhancer transition" and regulation of gene expression through epigenetic mechanisms .

How can CRISPR-based approaches be integrated with PAX9 antibody detection for functional genomics studies?

CRISPR-based approaches can be powerfully integrated with PAX9 antibody detection to conduct comprehensive functional genomics studies:

• CRISPR knockout validation: After generating PAX9 knockout cell lines using CRISPR-Cas9 (similar to the genome-wide CRISPR screening approach described in the research literature), researchers can use the PAX9 antibody in Western blot or immunofluorescence to confirm complete loss of protein expression .

• CRISPR knock-in of tagged PAX9: Researchers can use CRISPR to introduce epitope tags or fluorescent proteins to the endogenous PAX9 gene and verify correct tagging using the PAX9 antibody against the native protein.

• Domain-specific mutations: CRISPR-mediated homology-directed repair can be used to introduce specific mutations in PAX9 (similar to those described in the research: R26W, R47P, I56N, and A108P) followed by immunodetection to assess protein expression and localization changes .

• CRISPRi/CRISPRa with PAX9 antibody readout: Researchers can use CRISPRi to repress or CRISPRa to activate PAX9 expression or its target genes, then use the PAX9 antibody to quantify protein level changes and correlate with phenotypic outcomes.

• CRISPR screens for PAX9 modulators: Similar to the genome-wide CRISPR library screening approach described in the literature, researchers can conduct screens for genes that modulate PAX9 expression or activity, using the PAX9 antibody as a readout in high-content imaging or flow cytometry .

This integration of CRISPR technology with PAX9 antibody detection enables precise genetic manipulation with protein-level validation, advancing our understanding of PAX9 biology and regulatory networks.

How should researchers address non-specific binding or background issues when using PAX9 antibody (FITC conjugated)?

When encountering non-specific binding or high background with FITC-conjugated PAX9 antibody, researchers should implement the following systematic troubleshooting approaches:

• Optimize blocking conditions: Increase the concentration of blocking protein (BSA or normal serum) to 3-5% and extend blocking time to 1-2 hours at room temperature.

• Validate antibody specificity: Use the specific blocking peptide (Catalog # AAP34270) in a competition assay to confirm that observed signals are specific to PAX9 .

• Address autofluorescence: For tissues with high autofluorescence, pretreat sections with sodium borohydride (1mg/ml in PBS) for 10 minutes or use commercial autofluorescence quenching reagents.

• Optimize fixation protocols: Overfixation can mask epitopes while underfixation may compromise tissue morphology. For the N-terminal region of PAX9, mild fixation conditions are generally preferred.

• Titrate antibody concentration: Test a range of dilutions around the recommended 0.5 mg/ml concentration to find the optimal signal-to-noise ratio for your specific application .

• Include additional wash steps: After antibody incubation, increase the number and duration of wash steps with 0.1% Tween-20 in PBS to remove unbound antibody.

• Use appropriate negative controls: Always include a FITC-conjugated isotype control and ideally a PAX9-negative tissue section to distinguish true signal from non-specific binding.

For particularly challenging applications, consider using amplification methods such as tyramide signal amplification that can allow for more dilute antibody concentrations while maintaining signal intensity.

Why might researchers observe discrepancies between PAX9 protein detection and mRNA expression levels?

Discrepancies between PAX9 protein detection using antibodies and mRNA expression levels detected by PCR can occur for several biological and technical reasons:

• Post-transcriptional regulation: Research on PAX9 has shown that mRNA stability can be affected by mutations or cellular conditions. As demonstrated in mRNA stability studies using actinomycin D treatment, wild-type and mutant PAX9 mRNA may degrade at different rates, leading to differences between mRNA and protein levels .

• Protein stability differences: Some PAX9 mutations, particularly frameshift mutations (like 146delC, 185_189dup, 256_262dup, and 592delC) can affect protein stability without necessarily altering mRNA levels .

• Epitope accessibility issues: The N-terminal region epitope recognized by the antibody may be masked in certain protein conformations or protein-protein interactions, resulting in underdetection of the protein despite abundant mRNA.

• Differential detection sensitivity: qRT-PCR can detect very low copy numbers of mRNA transcripts, sometimes below the detection threshold of antibody-based protein detection methods.

• Temporal differences: Due to delays between transcription and translation, plus differences in mRNA and protein half-lives, there may be temporal disconnects between peak mRNA and peak protein expression.

To address these discrepancies, researchers should consider conducting time-course experiments, using protein degradation inhibitors (like MG132), and employing multiple detection methods (Western blot, immunofluorescence) alongside mRNA quantification to build a comprehensive understanding of PAX9 expression regulation.

How should results be interpreted when studying PAX9 mutants using immunodetection methods?

When studying PAX9 mutants using immunodetection methods, researchers should consider the following interpretative frameworks:

• Subcellular localization analysis: Wild-type PAX9 typically demonstrates strong nuclear localization consistent with its function as a transcription factor . Mutations may disrupt nuclear localization signals or protein folding, resulting in cytoplasmic accumulation or aggregation. Quantitative analysis of nuclear-to-cytoplasmic signal ratios across multiple cells provides objective assessment of localization defects.

• Expression level interpretation: Some mutations may affect protein stability. Research has shown that frameshift mutations can lead to protein degradation despite normal mRNA levels . When quantifying Western blot or immunofluorescence intensity, normalize to appropriate loading controls and compare across multiple experiments.

• Functional domain impact assessment: The N-terminal region contains part of the paired box domain critical for DNA binding. Mutations in this region (such as R26W, R47P, I56N, and A108P) have been shown to disrupt DNA binding capacity in gel shift assays . Correlate immunodetection results with functional assays like luciferase reporter activation.

• Co-localization with interaction partners: Since PAX9 interacts with the NuRD complex to regulate gene expression , co-localization studies using the PAX9 antibody alongside antibodies against NuRD components can reveal whether mutations disrupt these protein-protein interactions.

• Correlation with phenotypic outcomes: In the context of tooth agenesis or cancer studies, correlate immunodetection results with phenotypic data to establish genotype-phenotype relationships.

This multi-dimensional interpretation approach allows researchers to gain comprehensive insights into how specific mutations affect PAX9 protein function, potentially informing therapeutic strategies for PAX9-related disorders.

How can PAX9 antibody be utilized in cancer research and what are the key considerations?

PAX9 antibody offers valuable applications in cancer research based on recent findings about PAX9's role in cancer progression and epigenetic regulation:

• Tumor classification and biomarker development: PAX9 expression patterns detected by immunohistochemistry can help classify tumor subtypes, particularly in small cell lung cancer (SCLC) where PAX9 has been identified as a potential oncogenic driver . Researchers should use standardized scoring systems to quantify nuclear PAX9 staining intensity and percentage of positive cells.

• Epigenetic enhancer regulation studies: PAX9 has been shown to interact with the NuRD complex at enhancers to repress nearby gene expression, which can be reversed by HDAC inhibition . Researchers can use PAX9 antibody in ChIP-seq studies to map enhancer binding sites across different cancer types and correlate with gene expression data.

• Therapeutic target validation: Since pharmacologic HDAC inhibition can reverse PAX9/NuRD-mediated gene repression, researchers can use PAX9 antibody to monitor protein expression and localization changes following drug treatment .

• Cancer progression mechanisms: PAX9 antibody can be used to detect changes in protein expression during cancer progression, particularly focusing on the "primed-active enhancer transition" that results in altered expression of neural differentiation and tumor-suppressive genes .

• Patient-derived xenograft (PDX) models: When establishing PDX models of PAX9-expressing tumors, researchers should use the antibody to confirm that PAX9 expression is maintained in the xenograft, preserving this aspect of the original tumor biology.

Key considerations include using multiple detection methods (IF, IHC, Western blot) to confirm findings, correlating with chromatin state mapping, and integrating with functional genomics approaches like CRISPR screening to identify synthetic lethal interactions with PAX9.

What advanced imaging techniques can be combined with PAX9 antibody (FITC conjugated) for developmental biology studies?

For developmental biology studies investigating PAX9 expression patterns and function, several advanced imaging techniques can be effectively combined with FITC-conjugated PAX9 antibody:

• 3D confocal microscopy: This enables visualization of PAX9 expression patterns within the three-dimensional context of developing structures such as tooth primordia, thymus, and skeletal elements .

• Live tissue imaging: Using explant cultures of PAX9-expressing tissues, researchers can perform time-lapse imaging to track dynamic changes in PAX9 expression during developmental processes.

• Light-sheet fluorescence microscopy (LSFM): This technique offers reduced photobleaching and phototoxicity compared to confocal microscopy, allowing for long-term imaging of PAX9 expression in developing tissues with minimal damage.

• Super-resolution microscopy: Techniques such as Stimulated Emission Depletion (STED) or Structured Illumination Microscopy (SIM) can resolve PAX9 localization within subnuclear structures, potentially revealing associations with specific chromatin domains.

• Spatial transcriptomics integration: Combining immunofluorescence detection of PAX9 protein with spatial transcriptomics techniques allows researchers to correlate protein localization with transcriptional profiles across developmental tissues.

• Correlative light and electron microscopy (CLEM): This approach enables researchers to detect PAX9 by fluorescence microscopy and then examine the ultrastructural context of PAX9-positive cells by electron microscopy.

When implementing these techniques, researchers should optimize fixation protocols to preserve both epitope accessibility and tissue architecture, and consider using spectral unmixing to distinguish FITC signal from tissue autofluorescence, particularly in developing tooth structures.

How can researchers quantitatively assess PAX9 protein-protein interactions using the FITC-conjugated antibody?

For quantitative assessment of PAX9 protein-protein interactions using FITC-conjugated antibody, researchers can employ several advanced techniques:

• Förster Resonance Energy Transfer (FRET): When PAX9-FITC antibody (donor) is in close proximity to an acceptor fluorophore-conjugated antibody targeting a potential interaction partner (such as components of the NuRD complex), FRET can occur. This can be measured by acceptor photobleaching or fluorescence lifetime imaging microscopy (FLIM), providing quantitative data on protein proximity in situ.

• Proximity Ligation Assay (PLA): This technique can detect interactions between PAX9 and potential partners when they are within 40nm of each other, generating quantifiable fluorescent spots. The FITC-conjugated PAX9 antibody would be used alongside an unconjugated antibody against the interaction partner, followed by appropriate PLA probes.

• Quantitative Co-localization Analysis: Using high-resolution confocal microscopy, researchers can perform pixel-based co-localization analysis between PAX9-FITC and potential interaction partners, calculating Pearson's correlation coefficient or Manders' overlap coefficient.

• Fluorescence Cross-Correlation Spectroscopy (FCCS): This technique can detect complex formation between PAX9 and other proteins by measuring coupled diffusion in solution or in live cells, requiring the PAX9-FITC antibody and a differently labeled antibody against the potential interaction partner.

• Quantitative Immunoprecipitation: Using the PAX9 antibody for immunoprecipitation followed by quantitative Western blotting can provide stoichiometric information about protein complexes. The immunoprecipitation protocol should be optimized based on published methods .

These approaches provide complementary information about PAX9 interactions, from in situ detection to biochemical quantification, enabling researchers to build comprehensive models of PAX9 regulatory complexes in different biological contexts.