PEX11A Antibody, FITC conjugated

What is the biological function of PEX11A in cellular systems?

PEX11A functions as a key regulator in peroxisomal biogenesis, specifically involved in peroxisomal proliferation and membrane dynamics. This 28 kDa peroxisomal integral membrane protein mediates the binding of coatomer proteins to the peroxisomal membrane and regulates peroxisome division . PEX11A is highly expressed in kidney and liver tissues, where peroxisomal activity is particularly important for metabolic functions . Research has demonstrated that PEX11A deficiency significantly impairs peroxisome elongation, leading to reduction of functional peroxisomes, decreased fatty acid oxidation capacity, and potential development of steatosis . Understanding these functions provides crucial context for experimental design when using PEX11A antibodies.

What are the typical applications for PEX11A antibodies in research settings?

PEX11A antibodies are primarily utilized in Western Blot (WB) applications with validated dilution ranges of 1:1000-1:4000 or 1:500-1:2000 depending on the specific antibody preparation . While conventional antibodies are employed in WB and ELISA techniques, FITC-conjugated variants extend application possibilities to include direct fluorescence microscopy, flow cytometry, and immunohistochemistry without requiring secondary antibody incubation steps. When designing experiments, researchers should consider that validated reactivity typically includes human and mouse samples, with predicted homology for other species including cow (79%), dog (79%), and pig (77%) . Methodologically, optimization is sample-dependent and requires titration within each testing system to achieve optimal signal-to-noise ratio.

How should researchers properly store and handle PEX11A antibodies to maintain integrity?

For optimal preservation of antibody function, PEX11A antibodies should be stored at -20°C where they typically remain stable for one year after shipment . For short-term use (up to one week), storage at 2-8°C is acceptable . Most preparations are supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability . To prevent activity loss through repeated freeze-thaw cycles, researchers should divide the antibody into small working aliquots upon receipt . When preparing dilutions for experiments, use fresh buffer systems and handle the antibody at appropriate temperatures to prevent protein denaturation. Monitoring performance through positive controls in each experimental run allows verification of continued antibody functionality.

What optimization steps are necessary when using FITC-conjugated PEX11A antibodies for immunofluorescence?

When implementing FITC-conjugated PEX11A antibodies for immunofluorescence applications, several methodological optimizations are essential. Begin with a fixation comparison study evaluating paraformaldehyde (4%) versus methanol fixation to determine which better preserves the peroxisomal membrane structure while maintaining epitope accessibility. Permeabilization requires careful optimization with detergents (0.1-0.5% Triton X-100 or 0.1% saponin) to allow antibody access to membrane-associated PEX11A without disrupting peroxisomal integrity.

Due to FITC's sensitivity to photobleaching, minimize exposure to light during processing, mount samples using anti-fade reagents containing DAPI for nuclear counterstaining, and perform image acquisition promptly using appropriate excitation (490nm) and emission (525nm) filter sets.

How can researchers confirm the specificity of PEX11A antibody binding in their experimental systems?

• Western blot analysis using tissue/cell lysates known to express PEX11A (kidney or liver samples are optimal) to confirm detection at the expected molecular weight of 28 kDa .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (portions of the PEX11A sequence used for immunization, such as amino acids 110-180 of human PEX11A ) before application to samples, which should substantially reduce or eliminate specific signals.

• Positive and negative control tissues: Compare staining patterns between tissues known to have high expression (kidney, liver) versus those with minimal expression.

• Genetic validation: Employ PEX11A knockout or knockdown systems (CRISPR-Cas9 or siRNA) as definitive negative controls.

• Co-localization studies: For FITC-conjugated antibodies, perform double-labeling with established peroxisomal markers (catalase or PMP70) to confirm expected organelle localization pattern.

• Cross-reactivity assessment: Test the antibody against related family members (PEX11B, PEX11G) to ensure specificity within the PEX11 protein family.

How can PEX11A antibodies be used to investigate peroxisome dynamics in live cell imaging?

While traditional FITC-conjugated antibodies cannot penetrate intact cell membranes, advanced techniques enable investigation of peroxisome dynamics using PEX11A antibodies. For live cell applications, researchers should consider:

• Generation of chimeric fluorescent protein constructs: Create PEX11A-GFP fusion proteins through molecular cloning that can be transiently transfected or stably expressed in cell lines. These allow direct visualization of PEX11A localization and trafficking without antibody staining.

• Microinjection approach: For specialized applications, directly introduce small amounts of FITC-conjugated PEX11A antibodies into living cells using microinjection techniques, monitoring peroxisome dynamics in real-time using confocal microscopy.

• Permeabilization-based methods: Employ mild permeabilization techniques (0.01% digitonin) that maintain cell viability while allowing limited antibody access for short-term live imaging.

• Correlative microscopy workflow: Implement a hybrid approach where live cell imaging with peroxisome-targeted fluorescent proteins is followed by fixation and immunostaining with PEX11A antibodies, allowing correlation between dynamic behaviors and PEX11A distribution.

These methodological approaches provide complementary data to fixed-cell immunofluorescence, revealing aspects of peroxisome membrane dynamics and PEX11A's role in organelle remodeling that would otherwise remain obscured.

What strategies can address potential cross-reactivity issues when studying PEX11A in complex tissue samples?

Cross-reactivity represents a significant challenge when investigating PEX11A in complex tissue environments. Advanced researchers should implement these methodological strategies:

• Sequential immunoprecipitation: First deplete samples of potential cross-reactive proteins using antibodies against related peroxins (particularly PEX11B and PEX11G) before PEX11A immunoprecipitation or detection.

• Signal verification through multiple antibody approach: Utilize antibodies recognizing different epitopes within PEX11A protein (N-terminal vs. C-terminal regions) to confirm consistent localization patterns.

• Bioinformatic sequence analysis: Conduct thorough in silico analysis of the immunogen sequence used to generate the antibody (such as amino acids 110-180 of human PEX11A ) against the proteome of the species under investigation to identify potential cross-reactive proteins based on sequence homology.

• Combinatorial labeling: Implement multi-color labeling schemes where PEX11A (FITC channel) is co-detected with other peroxisomal markers using spectrally distinct fluorophores, confirming appropriate organelle localization.

• Absorption controls: Pre-absorb the PEX11A antibody with recombinant protein from related family members to reduce potential cross-reactivity while maintaining specific binding to the target antigen.

These approaches significantly enhance confidence in the specificity of observed signals in complex biological systems such as tissue sections or primary cell cultures.

How should researchers interpret discrepancies in PEX11A localization between immunofluorescence and biochemical fractionation studies?

Discrepancies between different methodological approaches are common in peroxisome biology research. When investigating PEX11A, researchers may observe differences between FITC-based immunofluorescence localization and biochemical fractionation results due to several factors:

• Fixation artifacts: Aldehyde fixatives can alter membrane protein epitopes differently than organic solvents, potentially affecting antibody recognition. Compare multiple fixation methods to determine if discrepancies are fixation-dependent.

• Extraction conditions: The ionic strength, detergent type, and pH used in fractionation buffers may differentially solubilize PEX11A from peroxisomal membranes. Systematic buffer optimization should be performed.

• Peroxisome fragility: Peroxisomes are sensitive to homogenization conditions, potentially releasing membrane proteins differently than observed in intact cells. Gentle fractionation methods may better preserve in vivo associations.

• Population heterogeneity: PEX11A may distribute differently across peroxisome subpopulations that may not be equally represented in biochemical versus microscopy studies. Single-organelle analysis techniques can address this issue.

• Dynamic localization: PEX11A shuttles between different subcellular compartments depending on cellular state. Time-course studies combining both approaches can reconcile apparent discrepancies.

What considerations are important when designing experiments to study the role of PEX11A in models of metabolic disease?

When investigating PEX11A in metabolic disease models, experimental design must account for several critical factors:

• Tissue-specific regulation: PEX11A exhibits tissue-selective regulation through PPARα in liver and PPARγ in adipose tissue via a shared peroxisome proliferator response element (PPRE) . Experimental designs must account for this differential regulation when selecting appropriate tissues and experimental conditions.

• Nutritional status effects: Fasting/feeding cycles significantly impact peroxisome biology and PEX11A expression. Standardize nutritional status across experimental groups and explicitly report feeding conditions.

• Appropriate controls for PPAR activators: When using PPAR agonists or studying conditions with altered lipid metabolism, include both wild-type and PPAR-null models to distinguish direct versus PPAR-mediated effects on PEX11A expression .

• Age and sex considerations: Peroxisomal function shows significant age and sex dimorphism. Age-matched controls and sex-stratified analysis should be standard practice.

• Quantification methodology:

These methodological considerations ensure robust data generation when investigating PEX11A's role in metabolic dysfunction, potentially revealing new insights into conditions like non-alcoholic fatty liver disease where peroxisomal dysfunction may contribute to pathogenesis.

How can dual-color imaging with PEX11A antibodies advance understanding of peroxisome-organelle contacts?

Peroxisome-organelle contact sites represent an emerging field in cell biology. FITC-conjugated PEX11A antibodies can be strategically employed in multi-color imaging approaches to investigate these dynamic interfaces:

• Proximity analysis methodology: Combine FITC-conjugated PEX11A antibodies with antibodies against organelle markers (using spectrally distinct fluorophores like Texas Red or Cy5) for mitochondria (TOM20), endoplasmic reticulum (Sec61β), or lipid droplets (PLIN1). Analyze co-localization using super-resolution techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy to resolve structures below the diffraction limit.

• Expansion microscopy approach: Apply physical expansion of fixed samples to increase effective resolution of conventional confocal microscopy when visualizing PEX11A and interacting organelles.

• Sequential imaging workflow: Implement a correlative light-electron microscopy approach where fluorescence imaging of PEX11A-FITC is followed by electron microscopy of the same sample area, providing ultrastructural context to fluorescence observations.

• FRET-based methodologies: For specialized applications, design fluorescence resonance energy transfer (FRET) pairs between PEX11A and candidate proteins on adjacent organelles to detect proximity within the 10nm range, substantially below optical resolution limits.

These advanced imaging approaches can reveal how PEX11A may function at peroxisome-organelle interfaces, potentially identifying novel roles beyond peroxisome division in metabolic coordination between organelles.

What strategies can researchers employ to study post-translational modifications of PEX11A using immunological approaches?

Post-translational modifications (PTMs) critically regulate PEX11A function, yet remain challenging to study. Advanced methodological approaches include:

• Modification-specific antibody development: Generate or source antibodies specifically recognizing known PTMs of PEX11A, such as phosphorylated serine residues or ubiquitination sites. Validate specificity using in vitro modified recombinant protein.

• Two-dimensional immunoblotting approach: Separate proteins first by isoelectric point (revealing charge changes from PTMs) then by molecular weight before probing with PEX11A antibodies to resolve modified forms.

• Immunoprecipitation coupled with mass spectrometry:

• Comparison of native versus denatured immunoprecipitation: Some PTM-specific epitopes may be masked in the native conformation of PEX11A. Compare native versus denatured immunoprecipitation conditions to maximize detection of modified forms.

• Stimulation/inhibition experiments: Implement pharmacological modulators of kinases, phosphatases, or ubiquitin pathway components coupled with immunodetection to determine the regulatory enzymes controlling PEX11A modifications.

These methodological approaches provide complementary information about the complex post-translational regulation of PEX11A, potentially revealing novel intervention points for modulating peroxisome dynamics in disease states.