PEX11A Antibody

What is PEX11A and why is it significant for metabolic research?

PEX11A (peroxisomal biogenesis factor 11 alpha) is a 28 kDa integral membrane protein that plays a crucial role in peroxisome elongation and proliferation . Peroxisomes are ubiquitous single-membrane organelles essential for cellular metabolism, particularly fatty acid oxidation. PEX11A is highly expressed in metabolically active tissues such as kidney and liver .

The significance of PEX11A in metabolic research stems from evidence that PEX11A deficiency impairs peroxisome elongation, leading to reduced functional peroxisomes and decreased fatty acid oxidation capacity . This ultimately contributes to steatosis (abnormal fat accumulation in tissues) and metabolic dysfunction. In mouse models, PEX11A deficiency results in adiposity, dyslipidemia, and impaired glucose tolerance, making it an important target for metabolic disease research .

What applications are PEX11A antibodies validated for?

Based on current documentation, commercial PEX11A antibodies are primarily validated for Western Blot (WB) applications . The Proteintech antibody (15481-1-AP) is additionally validated for ELISA applications . When designing experiments, researchers should consider the following application-specific parameters:

While these applications represent validated uses, researchers should perform additional validation when applying these antibodies to new experimental contexts or sample types .

How should PEX11A antibodies be stored to maintain optimal activity?

Proper storage is critical for antibody performance. For PEX11A antibodies, the following storage conditions are recommended:

• Short-term storage (up to 2 weeks): Refrigerate at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

Most commercial PEX11A antibodies are supplied in PBS buffer with stabilizing agents. For example:

• Proteintech's antibody (15481-1-AP) is supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Biorbyt's antibody (orb579770) is supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose

Antibody aliquoting is recommended to minimize freeze-thaw cycles, which can degrade antibody performance over time. The typical shelf life for these antibodies is approximately 12 months from the date of receipt when stored properly .

What species reactivity has been demonstrated for commercial PEX11A antibodies?

Understanding the species cross-reactivity of PEX11A antibodies is essential for experimental planning. Current commercial antibodies show the following reactivity profiles:

When working with species not listed as confirmed, preliminary validation experiments are strongly recommended to verify antibody specificity and sensitivity in your experimental system .

How does PEX11A deficiency affect lipid metabolism, and what can researchers learn from PEX11A-knockout models?

Studies using Pex11a knockout mice (Pex11a⁻/⁻) have revealed significant metabolic phenotypes that researchers should consider when designing PEX11A-related experiments:

• Adiposity and Body Composition Changes: Pex11a⁻/⁻ mice exhibit increased fat mass and decreased skeletal muscle mass, particularly when challenged with a high-fat diet (HFD) .

• Dyslipidemia Profile: These mice show elevated cholesterol levels and accumulation of very long- and long-chain fatty acids (C16:0-C24:0) in serum, with particularly significant increases in palmitic acid (C16:0) and stearic acid (C18:0) .

• Glucose Metabolism Impairment: Pex11a⁻/⁻ mice display higher fasting glucose levels, elevated hemoglobin A1c, impaired glucose tolerance, and reduced insulin sensitivity .

• Oxygen Consumption and Metabolic Rate: These mice consume less oxygen, indicating decreased fatty acid oxidation capacity, which aligns with the observed lipid accumulation phenotype .

For researchers studying metabolic diseases, these models provide valuable insights into the role of peroxisomal function in lipid homeostasis. When designing experiments with these models, researchers should include comprehensive metabolic phenotyping including:

• Body composition analysis

• Glucose and insulin tolerance tests

• Lipid profiling (particularly very long-chain fatty acids)

• Metabolic rate measurements

• Tissue-specific analyses of peroxisome abundance and function

What methodological approaches should be used to validate PEX11A antibody specificity?

Rigorous validation of antibody specificity is essential for reliable research outcomes. For PEX11A antibodies, researchers should implement the following validation strategies:

• Positive and Negative Controls:

  • Positive tissue controls: Use tissues with known high PEX11A expression (kidney, liver, adipose tissue)

  • Negative controls: Include PEX11A knockout samples when available, or tissues with minimal expression

  • Competing peptide assays: Pre-incubation with the immunizing peptide should abolish specific signal

• Multiple Detection Methods:

  • Compare protein detection using antibodies raised against different epitopes of PEX11A

  • Correlate protein detection with mRNA expression data

  • Consider orthogonal techniques such as mass spectrometry for protein identification

• Molecular Weight Verification:

  • Confirm that the detected band appears at the expected molecular weight (28 kDa for PEX11A)

  • Be aware of potential post-translational modifications that might alter apparent molecular weight

• Knockdown/Overexpression Validation:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of PEX11A and verify reduced signal

  • Overexpress tagged PEX11A and confirm co-detection with anti-tag and anti-PEX11A antibodies

These validation approaches should be documented and reported in publications to enhance reproducibility and reliability of research findings.

How can researchers distinguish between PEX11 family members (PEX11A, PEX11B, PEX11G) in experimental systems?

Distinguishing between PEX11 family members presents a significant challenge due to their structural similarities. Researchers should consider these methodological approaches:

• Antibody Selection and Validation:

  • Use antibodies generated against unique regions of each PEX11 protein

  • The middle region of PEX11A (e.g., the sequence MKRVTCDRAKKEKSASQDPLWFSVAEEETEWLQSFLLLLFRSLKQHPPLL) appears to be targeted in several commercial antibodies

  • Perform extensive cross-reactivity testing against all PEX11 family members

• Expression Pattern Analysis:

  • Leverage known tissue-specific expression patterns (PEX11A is prominently expressed in adipose tissue, liver, kidney, heart, gastrocnemius, and brain)

  • Use quantitative PCR to establish relative expression levels of different PEX11 isoforms in your experimental system

• Genetic Approaches:

  • Use family member-specific siRNAs or CRISPR/Cas9 targeting to validate antibody specificity

  • Consider using tagged versions of PEX11 proteins in overexpression studies

• Western Blot Optimization:

  • Optimize SDS-PAGE conditions to maximize separation of PEX11 family members

  • Consider using 2D gel electrophoresis to separate based on both molecular weight and isoelectric point

These strategies will help ensure accurate identification and characterization of specific PEX11 family members in complex biological samples.

What are the optimal Western blot conditions for detecting PEX11A?

Western blot optimization is critical for sensitive and specific detection of PEX11A. Based on published protocols and commercial recommendations, researchers should consider:

• Sample Preparation:

  • For tissues with high peroxisome content (liver, kidney), standard RIPA buffer with protease inhibitors is sufficient

  • For adipose tissue, consider specialized extraction buffers to remove lipids that may interfere with protein separation

  • Include reducing agents (DTT or β-mercaptoethanol) in loading buffer to ensure proper denaturation

• Gel Electrophoresis Parameters:

  • Use 10-12% polyacrylamide gels for optimal resolution of the 28 kDa PEX11A protein

  • Load appropriate positive controls (mouse kidney tissue has been validated)

  • Consider gradient gels when analyzing multiple proteins with diverse molecular weights

• Transfer and Blocking Conditions:

  • PVDF membranes are recommended for optimal protein binding

  • Standard transfer buffers (Tris-glycine with 20% methanol) work well for PEX11A

  • Block with 5% non-fat dry milk or 3-5% BSA in TBST

• Antibody Incubation:

  • Primary antibody dilutions: 1:1000-1:4000 for Western blot applications

  • Incubate overnight at 4°C for optimal specific binding

  • Wash thoroughly with TBST before secondary antibody incubation

• Detection Considerations:

  • Both chemiluminescent and fluorescent detection methods are suitable

  • For quantitative analysis, fluorescent detection offers superior linearity

  • Exposure times should be optimized based on signal strength

By systematically optimizing these parameters, researchers can achieve reliable and reproducible detection of PEX11A in diverse experimental contexts.

How can researchers effectively study the relationship between PEX11A expression and peroxisome function?

To investigate the relationship between PEX11A expression and peroxisome function, researchers should implement a multi-faceted experimental approach:

• Peroxisome Morphology Analysis:

  • Immunofluorescence microscopy using PEX11A antibodies along with established peroxisomal markers (e.g., catalase, PMP70)

  • Electron microscopy to assess ultrastructural changes in peroxisome morphology

  • Live-cell imaging with fluorescently tagged peroxisomal proteins to monitor peroxisome dynamics

• Functional Assays:

  • Measure β-oxidation of very long-chain fatty acids (C16:0-C24:0) as this pathway is impaired in PEX11A deficiency

  • Assess oxygen consumption rates as a proxy for oxidative metabolism

  • Quantify peroxisome-specific metabolites using mass spectrometry

• Genetic Manipulation Approaches:

  • Create dose-dependent expression systems (inducible promoters) to correlate PEX11A levels with peroxisome abundance

  • Use Pex11a knockout models and rescue experiments to establish causality

  • Apply CRISPR/Cas9 genome editing to introduce specific mutations and assess their impact

• Tissue-Specific Analyses:

  • Compare PEX11A expression and peroxisome function across multiple tissues

  • Use tissue-specific conditional knockout models to assess tissue-autonomous effects

  • Consider metabolic challenge conditions (e.g., high-fat diet) to stress the peroxisomal system

This integrated approach will provide comprehensive insights into how PEX11A regulates peroxisome function in different physiological and pathological contexts.

What experimental approaches can be used to study PEX11A's role in dyslipidemia and obesity?

Building on findings from Pex11a⁻/⁻ mouse models, researchers can employ several approaches to investigate PEX11A's role in metabolic disorders:

• Metabolic Phenotyping:

  • Body composition analysis using techniques such as DEXA or MRI to quantify fat and lean mass

  • Comprehensive metabolic panel including glucose tolerance tests, insulin sensitivity assays, and lipid profiling

  • Indirect calorimetry to measure oxygen consumption, carbon dioxide production, and physical activity

• Lipid Analysis Techniques:

  • Gas chromatography-mass spectrometry (GC-MS) to profile fatty acids, particularly very long-chain and long-chain saturated fatty acids (C16:0-C24:0)

  • Lipidomics to characterize comprehensive changes in lipid species and metabolism

  • Histological assessment of lipid accumulation in tissues using Oil Red O or other lipid stains

• Molecular Mechanism Investigation:

  • Analyze expression of lipogenic genes and proteins (e.g., fatty acid synthase) in adipose tissue and liver

  • Investigate signaling pathways involved in insulin response and lipid metabolism

  • Examine transcriptional regulation of PEX11A in response to metabolic challenges

• Therapeutic Intervention Studies:

  • Test whether enhancing peroxisome function can rescue metabolic phenotypes in PEX11A-deficient models

  • Investigate diet modifications or pharmacological interventions that might bypass PEX11A-dependent peroxisomal functions

  • Explore the potential of PEX11A as a therapeutic target for metabolic disorders

By implementing these approaches, researchers can develop a comprehensive understanding of how PEX11A contributes to lipid homeostasis and how its dysfunction leads to metabolic disorders.

What are the recommended experimental controls when working with PEX11A antibodies in immunohistochemistry?

For immunohistochemical applications with PEX11A antibodies, researchers should implement rigorous controls:

• Positive and Negative Tissue Controls:

  • Positive controls: Include tissues with known high PEX11A expression (kidney, liver, adipose tissue)

  • Negative controls: Include tissues with minimal PEX11A expression or PEX11A-knockout tissues when available

• Technical Controls:

  • Omit primary antibody while maintaining all other steps to assess non-specific binding of secondary antibody

  • Perform peptide competition assays by pre-incubating the antibody with immunizing peptide

  • Include isotype controls using non-specific IgG from the same species as the primary antibody

• Sample Processing Considerations:

  • Optimize fixation conditions (typical protocols use citric acid solution for antigen retrieval)

  • Validate staining patterns across different fixation methods

  • Perform parallel staining with multiple antibodies targeting different epitopes of PEX11A

• Co-localization Studies:

  • Co-stain with established peroxisomal markers to confirm correct subcellular localization

  • Use fluorescent double-labeling to assess co-localization with other proteins of interest

  • Include F4/80 staining (or other appropriate markers) when studying inflammatory conditions

By systematically implementing these controls, researchers can confidently interpret immunohistochemical data involving PEX11A detection.

How can advanced technologies be integrated with PEX11A antibodies for comprehensive functional studies?

Integrating cutting-edge technologies with PEX11A antibodies can significantly enhance functional studies:

• Proximity Labeling Approaches:

  • BioID or APEX2 fusion proteins can identify proximal interacting partners of PEX11A in living cells

  • These methods can reveal the dynamic PEX11A interactome under different physiological conditions

• Super-Resolution Microscopy:

  • STED, STORM, or PALM microscopy can resolve sub-peroxisomal localization of PEX11A

  • These techniques enable visualization of PEX11A distribution during peroxisome elongation and division

• Proteomics Integration:

  • Immunoprecipitation coupled with mass spectrometry can identify PEX11A-interacting proteins

  • Quantitative proteomics can assess changes in the peroxisomal proteome in response to PEX11A manipulation

• Single-Cell Analysis:

  • Single-cell RNA-seq combined with protein detection can reveal heterogeneity in PEX11A expression

  • CyTOF or imaging mass cytometry can profile PEX11A in relation to multiple markers simultaneously

• Genome-Scale Screening:

  • CRISPR screens can identify genes that modify PEX11A-dependent phenotypes

  • Chemical genomics approaches can discover compounds that modulate PEX11A function or bypass PEX11A deficiency

These integrated approaches provide multidimensional insights into PEX11A function, moving beyond traditional antibody applications toward comprehensive understanding of its role in peroxisome biology and metabolic regulation.