PAX8 Antibody, HRP conjugated

What is PAX8 and why is it important in research?

PAX8 is a member of the paired box (PAX) family of transcription factors with a molecular weight of approximately 62 kDa. It functions as a transcription factor for thyroid-specific gene expression and maintains the functional differentiation of thyroid cells . PAX8 is also expressed in non-ciliated mucosal cells of fallopian tubes, renal tubules, and various cancers including ovarian, renal cell carcinoma, and nephroblastoma .

Its importance in research stems from its role as a biomarker in cancer diagnostics and its involvement in embryogenesis. PAX8 interactions with other proteins such as chromatin remodeling complexes make it a valuable target for studying transcriptional regulation mechanisms. Recent studies have demonstrated that PAX8 promotes angiogenesis in ovarian cancer, highlighting its potential as a therapeutic target .

What are the optimal applications for HRP-conjugated PAX8 antibodies?

HRP-conjugated PAX8 antibodies are particularly valuable for:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) for direct visualization without secondary antibody steps

• Western blot analysis with enhanced sensitivity and reduced background

• Flow cytometry applications requiring minimal protocol steps

• CyTOF (mass cytometry) experiments

• Protein array studies

The HRP conjugation eliminates the need for secondary antibody incubation, reducing protocol time and potential non-specific binding. This is especially advantageous when working with limited tissue samples or when performing multiplex staining procedures.

What is the subcellular localization pattern of PAX8 and how can it be effectively visualized?

PAX8 exhibits predominantly nuclear localization, consistent with its function as a transcription factor . This localization pattern can be effectively visualized using immunofluorescence or immunohistochemistry techniques.

For optimal visualization:

• Use appropriate antigen retrieval methods (heat-mediated antigen retrieval with Tris/EDTA buffer pH 9 has shown good results)

• Apply the HRP-conjugated PAX8 antibody at the recommended dilution (typically 1/500-1/1000 for IHC-P)

• For immunofluorescence, counterstain nuclei with DAPI to confirm nuclear localization

• For IHC-P with HRP-conjugated antibodies, develop with DAB substrate and counterstain with hematoxylin

High-resolution immunofluorescence analysis can reveal the precise nuclear co-localization of PAX8 with other transcription factors such as SOX17, as demonstrated in fallopian tube secretory cell lines and high-grade serous ovarian carcinoma (HGSOC) cell lines .

How can I detect PAX8 protein-protein interactions using HRP-conjugated antibodies?

PAX8 forms large molecular complexes of approximately 600 kDa with various proteins involved in chromatin remodeling and transcriptional regulation . To study these interactions using HRP-conjugated PAX8 antibodies:

• Co-immunoprecipitation followed by Western blot analysis:

  • Prepare nuclear extracts from cells of interest

  • Perform immunoprecipitation using anti-PAX8 antibodies

  • Elute the complexes and analyze by Western blot for potential interacting partners

  • The HRP conjugation can provide direct detection of PAX8 in control blots

• Proximity Ligation Assay (PLA):

  • This technique enables visualization of protein-protein interactions in situ

  • Research has confirmed increased interaction between PAX8 and SOX17 in HGSOC cell lines using PLA

  • The interactions are predominantly localized in the nuclei, consistent with their roles as transcription factors

• Size-exclusion chromatography:

  • Analyze the purified PAX8 complex using size-exclusion chromatography to determine complex size

  • PAX8-containing complexes typically elute at approximately 600 kDa

  • Collect fractions and analyze by mass spectrometry to identify interacting proteins

What are the critical controls and validation steps for ChIP experiments using PAX8 antibodies?

Chromatin immunoprecipitation (ChIP) experiments with PAX8 antibodies require rigorous controls and validation:

• Input Controls:

  • Reserve a portion of the chromatin sample before immunoprecipitation

  • This represents the starting material and accounts for differences in DNA amounts

• Negative Controls:

  • Include immunoprecipitation with preimmune rabbit IgG (5 μg) alongside PAX8 antibody (5 μg)

  • This controls for non-specific binding of chromatin to antibodies or beads

• Positive Controls:

  • Target known PAX8 binding sites, such as the E2F1 promoter

  • Verify enrichment using PCR with primers specific to these regions

• Validation Steps:

  • Confirm antibody specificity by Western blot prior to ChIP

  • Verify protein expression in your cell model using immunofluorescence

  • Optimize chromatin fragmentation to obtain 200-500 bp fragments

  • Validate ChIP results with independent methodologies (e.g., reporter assays)

Recent advances include ChIC/CUT&RUN-seq applications, which offer improved signal-to-noise ratio compared to traditional ChIP-seq, particularly suitable for certain PAX8 antibody clones like EPR13510 .

How can I optimize PAX8 detection in ovarian cancer tissue samples?

Ovarian cancer exhibits significantly higher PAX8 expression compared to normal ovarian tissue, making PAX8 an important marker for high-grade serous ovarian carcinoma (HGSOC) . To optimize PAX8 detection in these samples:

• Tissue Processing:

  • Use formalin-fixed, paraffin-embedded (FFPE) sections of 4-5 μm thickness

  • Ensure proper fixation time (24 hours) to preserve antigenicity

• Antigen Retrieval:

  • Heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0) provides optimal results

  • Maintain temperature at 95-98°C for 20 minutes followed by cooling

• Antibody Selection and Dilution:

  • For IHC-P, use HRP-conjugated PAX8 antibodies at 1/500-1/1000 dilution

  • For double immunofluorescence (e.g., PAX8 and SOX17), optimize each antibody separately first

• Detection Systems:

  • For HRP-conjugated antibodies, use DAB substrate with optimized development time

  • Counterstain with hematoxylin for nuclear contrast

• Interpretation Guidelines:

  • PAX8 shows nuclear staining pattern in ovarian cancer cells

  • Expression is significantly higher in HGSOC compared to normal ovarian surface epithelium

  • Co-expression with SOX17 is commonly observed in fallopian tube secretory epithelial cells and HGSOC

Why might I observe non-specific background in Western blots using HRP-conjugated PAX8 antibodies?

Non-specific background is a common challenge when using HRP-conjugated antibodies in Western blot applications. To minimize this issue:

• Blocking Optimization:

  • Test different blocking agents (5% non-fat milk, 5% BSA, commercial blocking buffers)

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

• Antibody Dilution:

  • Use appropriate dilutions (1/1000-1/10000) of HRP-conjugated PAX8 antibodies

  • Prepare antibodies in fresh blocking buffer with 0.1% Tween-20

• Washing Protocol:

  • Increase washing time and number of washes (5-6 washes of 5-10 minutes each)

  • Use TBS-T (0.1% Tween-20) for washing

• Reducing Agents:

  • Ensure complete reduction of samples with fresh DTT or β-mercaptoethanol

  • This prevents non-specific binding to partially reduced proteins

• Detection Sensitivity:

  • Use highly sensitive ECL substrates appropriate for your expected signal intensity

  • Short exposure times can help minimize background while capturing specific signals

Western blot analysis has successfully detected PAX8 in various cell lines including HEK-293T and SK-OV-3 using anti-PAX8 antibodies at 1/10000 and 1/1000 dilutions respectively .

How can I differentiate between PAX8 isoforms in my experimental system?

PAX8 exists in multiple isoforms due to alternative splicing, which can complicate interpretation of experimental results. To differentiate between these isoforms:

• Antibody Selection:

  • Choose antibodies targeting specific regions of PAX8

  • Antibodies recognizing recombinant fragments within amino acids 1-250 can detect multiple isoforms

  • Antibodies targeting regions around amino acids 60-261 are commonly used for research applications

• Western Blot Resolution:

  • Use lower percentage SDS-PAGE gels (8-10%) for better separation of closely spaced bands

  • Extend running time to achieve maximum separation

  • Use precision plus protein standards for accurate molecular weight determination

• Mass Spectrometry Analysis:

  • For definitive isoform identification, consider mass spectrometry following immunoprecipitation

  • This approach can identify specific peptides unique to each isoform

• RT-PCR Analysis:

  • Design primers specific to different exon junctions to detect alternative splicing events

  • Validate PAX8 isoform expression at the mRNA level before protein analysis

Western blot analysis typically reveals PAX8 as a protein of approximately 62kDa, but closely related isoforms may appear as multiple bands in the 48-62kDa range .

How can I utilize PAX8 antibodies to study the molecular mechanisms of ovarian carcinogenesis?

PAX8 plays a crucial role in ovarian carcinogenesis, particularly in high-grade serous ovarian carcinoma (HGSOC). HRP-conjugated PAX8 antibodies can be utilized to explore this process through:

• Characterization of PAX8-dependent transcriptional networks:

  • Perform ChIP-seq to identify genome-wide PAX8 binding sites

  • Combine with RNA-seq after PAX8 depletion to identify direct transcriptional targets

  • This approach has revealed that PAX8 forms a complex with SOX17 and promotes angiogenesis in ovarian cancer

• Analysis of PAX8 protein complexes:

  • Use biochemical affinity-purification methods to isolate endogenous PAX8 protein complexes

  • Nuclear extracts from ovarian carcinoma cell lines (OVCAR4, KURAMOCHI, OVSAHO) and immortalized fallopian tube secretory epithelial cells can be compared

  • Size-exclusion chromatography and mass spectrometry analysis reveal PAX8 exists in approximately 600 kDa complexes with chromatin remodeling components

• Visualization of PAX8-SOX17 interactions:

  • Use proximity ligation assay (PLA) to directly visualize protein-protein interactions in situ

  • Increased interaction between PAX8 and SOX17 has been observed in HGSOC cell lines compared to normal cells

  • These interactions are predominantly localized in the nuclei

• Functional studies of PAX8 in carcinogenesis:

  • Deploy CRISPR/Cas9 gene editing to modulate PAX8 expression

  • Assess effects on cell proliferation, migration, invasion, and angiogenesis

  • Evaluate tumor growth in xenograft models following PAX8 modulation

What are the most effective protocols for multiplexed detection of PAX8 with other cancer biomarkers?

Multiplexed detection of PAX8 with other cancer biomarkers provides valuable diagnostic and research insights. Here are effective protocols:

• Multiplex Immunofluorescence:

  • Use directly conjugated primary antibodies with different fluorophores

  • For HRP-conjugated PAX8 antibodies, employ tyramide signal amplification (TSA) with spectrally distinct fluorophores

  • Include PAX8 with markers such as SOX17, WT1, and p53 for HGSOC characterization

  • Use spectral imaging and unmixing to resolve overlapping emission spectra

• Sequential Immunohistochemistry:

  • For HRP-conjugated antibodies on the same section:

    1. Perform first staining with PAX8-HRP and develop with DAB (brown)

    2. Strip or block remaining HRP activity

    3. Apply second HRP-conjugated antibody and develop with alternative chromogen (e.g., AEC, red)

    4. Counterstain and analyze

• Mass Cytometry (CyTOF):

  • HRP-conjugated PAX8 antibodies can be adapted for CyTOF applications

  • Combine with metal-tagged antibodies against other cancer biomarkers

  • This allows simultaneous detection of >40 markers at single-cell resolution

• Multiplexed Protein Arrays:

  • HRP-conjugated PAX8 antibodies can be used in protein array applications

  • Design arrays containing multiple cancer biomarkers

  • Quantify relative expression levels across different sample types

• Digital Spatial Profiling:

  • Combine PAX8 detection with geographical tissue mapping

  • Correlate PAX8 expression with other markers in specific microenvironments

  • This provides insights into tumor heterogeneity and microenvironment interactions

How might PAX8 antibodies contribute to developing targeted therapies for PAX8-dependent cancers?

PAX8 represents a promising therapeutic target for cancers where it plays a crucial role in tumorigenesis. HRP-conjugated PAX8 antibodies can facilitate research in developing targeted therapies through:

• Identifying druggable protein-protein interactions:

  • PAX8 forms complexes with chromatin remodeling proteins including CHD4, GATAD2A, MTA2, HDAC1, and RBBP4

  • These interactions differ quantitatively between normal and cancer cells

  • Targeting specific interactions may provide cancer-selective therapeutic strategies

• Evaluating PAX8 inhibition strategies:

  • Screen for small molecule inhibitors that disrupt PAX8 binding to DNA or protein partners

  • Use HRP-conjugated PAX8 antibodies in high-throughput screening assays

  • Validate hits with secondary assays including ChIP, co-IP, and cellular phenotypic assays

• Developing PAX8-targeted antibody-drug conjugates:

  • Explore internalizing antibodies against PAX8 for payload delivery

  • Test efficacy in PAX8-positive cancer models

  • Evaluate specificity using PAX8 knockout controls

• Precision medicine applications:

  • Stratify patients based on PAX8 expression patterns

  • Correlate PAX8 complex formation with treatment response

  • Develop companion diagnostics for PAX8-targeted therapies

PAX8's role in promoting angiogenesis in ovarian cancer suggests that targeting this transcription factor could have anti-angiogenic effects, potentially enhancing current anti-angiogenic therapies.

What methodological advances are needed to better understand PAX8 regulation in normal and cancer tissues?

Despite significant progress, several methodological advances are needed to fully understand PAX8 regulation:

• Single-cell analysis of PAX8 expression and function:

  • Develop protocols for single-cell ChIP-seq to map PAX8 binding in rare cell populations

  • Combine with single-cell RNA-seq to correlate binding with gene expression

  • This would reveal cell-to-cell heterogeneity in PAX8 function within tumors

• Improved protein-protein interaction detection methods:

  • Develop more sensitive techniques to detect transient interactions

  • Implement BioID or APEX proximity labeling with PAX8 to identify the complete interactome

  • Quantitative interactome analysis between normal and cancer states

• Live-cell imaging of PAX8 dynamics:

  • Develop approaches to visualize PAX8 binding and complex formation in living cells

  • Engineer cell lines expressing fluorescently tagged PAX8 at endogenous levels

  • Study the dynamics of PAX8 recruitment to target genes during differentiation or carcinogenesis

• Structural studies of PAX8 complexes:

  • Determine the crystal structure of PAX8 bound to DNA and protein partners

  • Use cryo-EM to resolve the structure of larger PAX8-containing complexes

  • This would facilitate structure-based drug design targeting PAX8

• Improved antibody technology:

  • Develop recombinant antibodies with enhanced specificity for different PAX8 isoforms

  • Create antibodies that selectively recognize post-translationally modified PAX8

  • Engineer bi-specific antibodies for improved multiplexed detection