PPARD Monoclonal Antibody

What is PPARD and why is it significant for research applications?

PPARD, also known as NR1C2, FAAR, and Nuclear receptor subfamily 1 group C member 2, is a member of the peroxisome proliferator-activated receptor (PPAR) family. It functions as a nuclear hormone receptor that binds peroxisome proliferators and regulates the size and number of peroxisomes produced by cells. PPARD mediates numerous biological processes and has been implicated in the development of several chronic diseases, including diabetes, obesity, atherosclerosis, and cancer. It serves as a potent inhibitor of ligand-induced transcription activity of PPAR alpha and PPAR gamma and may function as an integrator of transcription repression and nuclear receptor signaling .

Research interest in PPARD has intensified due to its ubiquitous expression with maximal levels in placenta and skeletal muscle, and its elevated expression in certain cancer types, particularly colorectal cancer cells. This elevation can be repressed by adenomatosis polyposis coli (APC), a tumor suppressor protein related to the APC/beta-catenin signaling pathway . PPARD monoclonal antibodies are therefore essential tools for investigating these mechanisms in basic and translational research settings.

What applications are PPARD monoclonal antibodies most commonly used for?

PPARD monoclonal antibodies are primarily used in immunohistochemistry (IHC) applications, where they enable visualization of PPARD expression patterns in tissue samples. Based on the available product information, PPARD antibodies have been validated for:

• Immunohistochemistry-paraffin (IHC-p): Used to detect PPARD in formalin-fixed, paraffin-embedded tissue sections

• Western Blot (WB): Multiple publications have reported successful use in detecting PPARD protein in cell and tissue lysates

• Immunofluorescence (IF): At least one publication has reported successful application for fluorescent detection of PPARD

• ELISA: Some antibodies have been validated for enzyme-linked immunosorbent assay applications

Different antibody clones (such as 1D7 and 2F9) have been optimized for specific applications, and researchers should select the appropriate clone based on their experimental requirements .

What are the optimal storage and handling conditions for PPARD monoclonal antibodies?

For long-term storage, PPARD monoclonal antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . For frequent use and short-term storage (up to one month), antibodies can be stored at 4°C to avoid repeated freeze-thaw cycles that may compromise antibody integrity and performance .

Most PPARD monoclonal antibodies are supplied in a buffer containing stabilizers:

• Typically in phosphate-buffered saline (PBS)

• With 50% glycerol as a cryoprotectant

• Including protein protectants (0.5% BSA or similar)

• Containing preservatives (0.02% sodium azide)

When handling the antibody, it's important to:

• Aliquot upon initial thawing to minimize freeze-thaw cycles

• Briefly centrifuge the vial before opening to collect all liquid at the bottom

• Avoid contamination by using clean pipette tips

• Return to appropriate storage conditions immediately after use

What are the recommended dilution ratios for different experimental applications?

Optimal dilution ratios for PPARD monoclonal antibodies vary by application and specific antibody clone. Based on the product information provided, the following dilutions are recommended as starting points:

For IHC applications:

• Elabscience E-AB-22199: 1:100-200

• Boster Bio M01557: 1:100-200

• Proteintech 10156-2-AP: 1:10-100

For Western Blot applications:

• Proteintech 10156-2-AP: 1:500-1000

These values provide initial guidelines, but the optimal working concentration should be determined by each researcher through titration experiments in their specific experimental system. Factors such as tissue type, fixation method, detection system, and incubation conditions can all influence the optimal antibody concentration .

How do I optimize PPARD antibody performance in challenging tissue samples?

When working with challenging tissue samples, several optimization strategies can improve PPARD detection:

• Antigen retrieval optimization: The search results indicate that for some PPARD antibodies, TE buffer pH 9.0 is recommended for antigen retrieval, though citrate buffer pH 6.0 can be used as an alternative . For tissues with high lipid content or dense extracellular matrix, extended retrieval times may be necessary.

• Signal amplification approaches: For tissues with low PPARD expression, consider employing:

  • Polymer-based detection systems

  • Tyramide signal amplification

  • Extended primary antibody incubation (overnight at 4°C)

• Background reduction techniques: To reduce non-specific binding:

  • Increase blocking time (2-3 hours)

  • Use a combination of different blocking agents (serum, BSA, casein)

  • Perform longer washing steps with 0.1% Tween-20 in PBS

  • Include a peroxidase quenching step (if using HRP-based detection)

  • Consider titrating the antibody to identify the optimal concentration that gives specific signal with minimal background

• Validation controls: Always include positive control tissues known to express PPARD (such as human brain, mouse heart tissue, mouse skeletal muscle tissue) and negative controls (primary antibody omission and/or isotype controls) .

How can I validate PPARD antibody specificity for my experimental system?

Rigorous validation of PPARD antibody specificity is crucial for reliable research outcomes. A comprehensive validation approach should include:

• Multiple detection methods: Confirm PPARD detection using at least two independent methods (e.g., IHC and Western blot) .

• Knockout/knockdown controls: The gold standard for antibody validation includes:

  • Testing the antibody in PPARD-knockout tissues/cells

  • Using PPARD-knockdown samples (siRNA or shRNA) compared to controls

  • Observing the expected reduction or absence of signal

• Blocking peptide experiments: Pre-incubate the antibody with a specific blocking peptide (recombinant PPARD protein) to demonstrate signal specificity. The search results mention that blocking peptides can be purchased to validate antibody specificity .

• Multiple antibody comparison: Test multiple PPARD antibodies with different epitopes (e.g., compare monoclonal antibodies from different clones like 1D7 and 2F9) .

• Molecular weight verification: In Western blot applications, verify that the detected band aligns with the expected molecular weight of PPARD (calculated MW: ~50 kDa, observed MW: ~54 kDa) .

What are the contradictory findings regarding PPARD's role in cancer, and how should researchers interpret these discrepancies?

The literature presents contradictory findings regarding PPARD's role in cancer, which researchers must carefully consider when designing experiments and interpreting results. The table in the search results demonstrates this complexity, showing that PPARD can exhibit both pro-tumorigenic and anti-tumorigenic effects depending on context .

Key contradictions include:

• Tumor growth effects:

  • Pro-tumorigenic: In multiple models including LLC1 tumors, SW480 cells, and Apc(Min/+) mice, PPARD agonists like GW501516 increased tumor growth .

  • Anti-tumorigenic: In azoxymethane-induced colon tumors, GW0742 (PPARD agonist) decreased tumor growth. In transgenic hepatitis B virus mice, GW0742 reduced hepatic tumor foci .

• Cancer type specificity:

  • Colorectal cancer: Some studies show PPARD promotes tumorigenesis, while others show anti-tumorigenic effects .

  • Melanoma: PPARD agonists (GW0742, GW501516) decreased proliferation in multiple melanoma cell lines .

  • Breast cancer: Mixed effects observed depending on cell line (inhibited proliferation in MCF-7 but not in MDA-MB-231) .

To address these discrepancies, researchers should:

• Carefully design experiments with appropriate positive and negative controls

• Consider the specific cellular context and cancer type

• Document experimental conditions thoroughly (cell density, passage number, reagent concentrations)

• Use multiple complementary approaches to validate findings

• Consider the role of PPARD in different cell populations within the tumor microenvironment

• Examine potential differences in downstream signaling pathways

How can PPARD antibodies be incorporated into multi-parameter analysis of signaling pathways?

PPARD functions within complex signaling networks, and multi-parameter analysis can provide deeper insights into its regulatory mechanisms. Advanced researchers can integrate PPARD antibody detection with other techniques:

• Multiplex immunofluorescence/immunohistochemistry:

  • Co-stain tissues for PPARD alongside other pathway components (e.g., APC, β-catenin)

  • Use spectral unmixing to resolve multiple fluorophores

  • Employ tyramide signal amplification for detecting low-abundance signaling proteins

  • Combine with nuclear markers to confirm PPARD nuclear localization

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions between PPARD and potential binding partners

  • Validate transcriptional complexes involving PPARD

  • Investigate post-translational modifications of PPARD

• ChIP-seq integration:

  • Combine PPARD protein detection with chromatin immunoprecipitation sequencing to map genomic binding sites

  • Correlate PPARD protein levels with target gene expression

  • Identify cell-type-specific regulatory mechanisms

• Phospho-protein analysis:

  • Examine the phosphorylation status of PPARD and downstream effectors

  • Correlate PPARD activation with signaling pathway activity

  • Study the impact of therapeutic agents on PPARD-dependent signaling

What methodological approaches can resolve conflicting data between PPARD protein expression and functional activity?

Researchers often encounter situations where PPARD protein levels do not directly correlate with its transcriptional activity. To address this complexity:

• Combine protein detection with functional assays:

  • Use luciferase reporter assays with PPARD-responsive elements

  • Measure expression of known PPARD target genes by qRT-PCR

  • Assess metabolic changes associated with PPARD activation (fatty acid oxidation, glucose utilization)

• Characterize post-translational modifications:

  • Use phospho-specific antibodies to detect activation status

  • Examine SUMOylation, ubiquitination, and acetylation patterns

  • Correlate modifications with transcriptional activity

• Analyze protein-protein interactions:

  • Investigate co-repressor/co-activator recruitment

  • Examine interactions with other nuclear receptors (PPARα, PPARγ)

  • Study competitive binding of ligands and antagonists

• Consider ligand availability and metabolism:

  • Measure endogenous PPARD ligand levels

  • Evaluate the impact of metabolic changes on PPARD activity

  • Assess the influence of inflammatory mediators on PPARD function

What are common pitfalls when using PPARD antibodies in immunohistochemistry, and how can they be addressed?

Common challenges when using PPARD antibodies in IHC include:

• High background staining:

  • Problem: Non-specific binding to other proteins or tissue components.

  • Solutions: Optimize blocking (use 3-5% BSA or normal serum matching the secondary antibody host), increase washing duration and frequency, titrate primary antibody concentration, and ensure proper antigen retrieval .

• Weak or absent staining:

  • Problem: Insufficient antigen detection despite PPARD expression.

  • Solutions: Verify antibody reactivity with your species of interest (human, mouse, rat confirmed for most antibodies), optimize antigen retrieval (try both citrate buffer pH 6.0 and TE buffer pH 9.0), increase antibody concentration, extend incubation time, or employ signal amplification systems .

• Variable staining intensity:

  • Problem: Inconsistent results between experiments or tissue regions.

  • Solutions: Standardize fixation protocols, control tissue processing time, use automated staining platforms if available, include internal positive controls, and normalize staining time and temperature.

• Nuclear versus cytoplasmic localization:

  • Problem: Unexpected subcellular localization pattern.

  • Solutions: Verify fixation quality (overfixation can mask nuclear antigens), optimize permeabilization, compare with literature reports of PPARD localization in your specific tissue/cell type, and consider using cell fractionation followed by Western blot to confirm localization .

How should researchers interpret PPARD expression data in the context of cancer heterogeneity?

Cancer heterogeneity presents significant challenges for interpreting PPARD expression data. Advanced analytical approaches include:

• Spatial heterogeneity analysis:

  • Systematic sampling across different tumor regions

  • Quantitative image analysis with spatial statistics

  • Correlation of PPARD expression with histopathological features (tumor grade, invasion front, necrotic areas)

• Single-cell approaches:

  • Integration with single-cell RNA sequencing data

  • Multiplex immunofluorescence to analyze co-expression patterns

  • Cell-type specific analysis (tumor cells vs. stromal/immune components)

• Temporal dynamics consideration:

  • Serial sampling during disease progression

  • Analysis of primary tumors versus metastases

  • Treatment-induced changes in PPARD expression

• Functional correlation:

  • Link expression patterns to proliferation markers (Ki-67)

  • Correlate with markers of metabolic activity

  • Associate with treatment resistance phenotypes

The conflicting data in the literature regarding PPARD's role in cancer (promoting or inhibiting tumor growth depending on context) highlights the importance of considering tumor heterogeneity in experimental design and data interpretation .

How can PPARD monoclonal antibodies be used to investigate metabolic reprogramming in cancer?

PPARD plays crucial roles in metabolic regulation, making its antibodies valuable tools for studying cancer metabolism:

• Metabolic pathway correlation:

  • Co-stain for PPARD and key metabolic enzymes (e.g., FASN, ACC, CPT1)

  • Correlate PPARD expression with glucose transporters (GLUTs) and lactate transporters (MCTs)

  • Examine relationship with mitochondrial markers in different cancer models

• Microenvironmental adaptation:

  • Analyze PPARD expression in hypoxic versus normoxic regions (co-stain with HIF-1α)

  • Correlate with nutrient stress markers

  • Investigate expression changes in response to metabolic inhibitors

• Therapeutic implications:

  • Monitor PPARD expression changes following metabolic-targeted therapies

  • Assess PPARD as a biomarker for response to metabolic interventions

  • Explore combinatorial approaches targeting PPARD and metabolic vulnerabilities

• Lipid metabolism integration:

  • Combine PPARD immunodetection with lipid droplet staining

  • Correlate with fatty acid oxidation capacity

  • Investigate membrane lipid composition changes

The search results indicate that PPARD has been studied in various cancer contexts with different metabolic profiles, including colorectal, prostate, breast, and lung cancers .

What are emerging techniques for studying PPARD protein-protein interactions in situ?

Advanced techniques for investigating PPARD protein interactions in their native context include:

• Proximity Ligation Assay (PLA):

  • Enables visualization of protein-protein interactions (<40 nm proximity)

  • Can detect interactions between PPARD and co-regulators in fixed cells/tissues

  • Provides spatial information about interaction sites within cells

  • Quantifiable by digital image analysis

• FRET/FLIM microscopy:

  • Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging

  • Requires fluorophore-conjugated antibodies or expression of fluorescent fusion proteins

  • Provides dynamic information about protein interactions

  • Can detect conformational changes in protein complexes

• In situ hybridization with immunodetection:

  • Correlate PPARD protein localization with target gene transcription

  • RNAscope technology combined with immunofluorescence

  • Spatial transcriptomics integration with protein detection

• Mass spectrometry imaging:

  • Emerging technique for spatial proteomics

  • Can map PPARD interaction networks across tissue regions

  • Combines with traditional immunohistochemistry for validation

These approaches can help resolve contradictions in the literature by revealing context-specific interactions that may explain differential PPARD functions in various cancer types and experimental models .

How does PPARD research interface with studies of other nuclear receptors in cancer biology?

PPARD belongs to the nuclear receptor superfamily, and understanding its relationship with other family members is critical:

• Cross-regulation with other PPARs:

  • PPARD inhibits ligand-induced transcription activity of PPARα and PPARγ

  • Differential expression patterns across tissues require careful antibody validation

  • Potential redundancy or antagonism requires controlled experimental approaches

• Shared co-regulator networks:

  • Many nuclear receptors compete for the same co-activators/co-repressors

  • PPARD-specific effects must be distinguished from general nuclear receptor signaling

  • Validation using multiple approaches (genetic, pharmacological, immunological)

• Ligand specificity considerations:

  • Cross-reactivity of synthetic ligands between PPAR subtypes

  • Natural ligands may have multiple targets

  • Combine ligand studies with specific antibody detection for pathway validation

• Therapeutic implications:

  • PPARD agonists and antagonists have been developed for metabolic diseases

  • Cancer applications require careful consideration of context-dependent effects

  • Combination approaches targeting multiple nuclear receptors may offer advantages

The table in the search results highlights the complexity of PPARD functions in cancer, showing both pro- and anti-tumorigenic effects depending on context, similar to the context-dependent roles observed with other nuclear receptors .

What methodological approaches can link PPARD protein detection with genomic and transcriptomic data?

Integrating protein-level data with genomic and transcriptomic information provides a comprehensive understanding of PPARD biology:

• ChIP-seq correlation:

  • Map PPARD genomic binding sites using ChIP-seq

  • Correlate binding patterns with protein expression in matching samples

  • Identify cell type-specific regulatory elements

• Multi-omic integration:

  • Perform integrated analysis of PPARD protein levels, mRNA expression, and target gene activation

  • Identify discordances that suggest post-transcriptional regulation

  • Reveal feedback mechanisms and regulatory circuits

• Single-cell multi-modal analysis:

  • Emerging technologies allow simultaneous detection of proteins and mRNAs in single cells

  • Reveals heterogeneity in PPARD expression and activity at cellular resolution

  • Identifies rare cell populations with unique PPARD regulation

• Patient-derived models:

  • Validate PPARD antibody performance in patient-derived xenografts and organoids

  • Correlate with genomic alterations in the same samples

  • Develop personalized approaches based on PPARD status

These integrative approaches can help resolve contradictions in the literature by revealing the molecular contexts that determine whether PPARD promotes or inhibits cancer progression in specific settings .