PEX11A Antibody, HRP conjugated

What is the functional significance of PEX11A in cellular metabolism?

PEX11A plays a crucial role in peroxisomal biogenesis and function. It is primarily involved in peroxisomal proliferation and regulation of peroxisome division, acting as a key mediator in binding coatomer proteins to the peroxisomal membrane . Research has shown that PEX11A deficiency significantly impairs peroxisome elongation, which leads to reduction of functional peroxisomes and consequently diminishes fatty acid oxidation capacity, potentially resulting in steatosis . As a 28 kDa membrane protein, PEX11A (also known as PMP28 in some contexts) is particularly abundant in metabolically active tissues such as kidney and liver, suggesting tissue-specific regulatory roles .

What applications are most appropriate for PEX11A antibodies?

Based on current validation data, PEX11A antibodies are primarily optimized for Western Blot (WB) applications, with some products also validated for ELISA . The recommended dilution ranges for Western Blot applications vary by manufacturer:

When using HRP-conjugated detection systems, it's essential to optimize antibody concentrations to balance specific signal versus background.

How should PEX11A antibodies be stored for optimal performance?

Proper storage is critical for maintaining antibody performance. The consensus recommendation across manufacturers is to store PEX11A antibodies at -20°C for long-term preservation . For short-term use (up to one week), storage at 2-8°C is acceptable . Most PEX11A antibodies are supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3-7.4 . To minimize freeze-thaw cycles that can degrade antibody quality, it's advisable to aliquot the antibody upon receipt . Small aliquots (10-20μL) are recommended, particularly for HRP-conjugated antibodies, as enzyme activity can deteriorate with repeated freezing and thawing.

What considerations are important when selecting between direct HRP-conjugated primary antibodies versus two-step detection systems?

When deciding between direct HRP-conjugated PEX11A antibodies and unconjugated primary antibodies with secondary HRP detection, researchers should consider several factors:

In tissues with high endogenous peroxidase activity (e.g., liver where PEX11A is highly expressed), thorough peroxidase quenching is particularly important when using HRP-conjugated detection systems . The most effective approach often depends on the specific research question, sample type, and required sensitivity.

How can PEX11A antibodies be validated for specificity in experimental models?

Rigorous validation is essential to ensure experimental reliability. For PEX11A antibodies, a comprehensive validation strategy should include:

• Positive controls: Using tissues known to express high levels of PEX11A (kidney, liver)

• Molecular weight verification: Confirming the observed molecular weight matches the expected 28 kDa size

• Knockout/knockdown validation: Testing antibody in PEX11A-deficient models to confirm signal absence

• Peptide competition assays: Pre-incubating antibody with immunizing peptide to demonstrate specificity

• Cross-reactivity assessment: Testing in multiple species to confirm reactivity claims (human, mouse, rat)

For researchers studying PEX11A in neural differentiation contexts, shRNA-mediated knockdown approaches have been demonstrated as effective for validation purposes . When using HRP-conjugated systems, include appropriate enzyme activity controls to distinguish between specificity issues and detection problems.

What methodological approaches can resolve inconsistent PEX11A detection in different cell types?

PEX11A expression varies significantly between tissues, with particularly high expression in kidney and liver . This heterogeneity can lead to detection challenges. To address inconsistent detection:

• Optimize protein extraction: For membrane proteins like PEX11A, standard RIPA buffer may be insufficient. Consider specialized membrane protein extraction protocols that include mild detergents.

• Adjust loading controls: Different tissues may require different loading controls due to variable expression of housekeeping proteins.

• Enhance epitope accessibility: For formaldehyde-fixed samples, antigen retrieval optimization is critical as PEX11A epitopes may be differentially masked:

• Species-specific considerations: Human PEX11A detection may require different conditions than mouse or rat samples, despite high sequence homology (human sequence corresponding to amino acids 110-180 is used as immunogen in some products) .

What strategies can resolve weak or absent PEX11A signal in Western blot applications?

When encountering detection challenges with PEX11A antibodies:

• Sample preparation optimization:

  • Ensure proper membrane protein extraction using specialized buffers

  • Avoid excessive heating which may cause PEX11A aggregation

  • Consider non-reducing conditions if disulfide bonds are critical for epitope structure

• Protocol modifications:

  • Increase primary antibody concentration (within manufacturer guidelines)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use PVDF membranes rather than nitrocellulose for potentially better protein retention

  • Optimize blocking conditions (5% BSA may be superior to non-fat milk for some antibodies)

• Signal enhancement approaches:

  • For HRP-conjugated systems, consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Increase exposure time during imaging (60s or longer as demonstrated effective in validation studies)

• Loading considerations:

  • Increase protein loading (25μg per lane shown effective in validation studies)

  • Confirm sample integrity with additional markers

How can researchers optimize immunohistochemistry protocols for PEX11A detection?

When adapting PEX11A antibodies for immunohistochemistry or immunofluorescence:

• Fixation optimization:

  • 4% paraformaldehyde is generally suitable but may require optimization

  • Duration of fixation should be minimized to preserve epitope accessibility

  • Consider mild permeabilization for accessing membrane epitopes

• Antibody considerations:

  • Begin with higher concentrations than used for Western blot

  • For HRP-conjugated detection, thorough peroxidase quenching is essential (3% H₂O₂ for 10 minutes)

  • When using avidin-biotin-HRP systems, block endogenous biotin in tissues like liver

• Signal development:

  • For HRP systems, DAB development time should be carefully optimized

  • Consider tyramide signal amplification for low abundance detection

  • For co-localization studies with peroxisomal markers, fluorescent secondary antibodies may provide better resolution than HRP

What approaches can minimize background in PEX11A immunostaining?

Background issues are common challenges when working with membrane proteins like PEX11A:

• Blocking optimization:

  • For HRP-conjugated systems, sequential blocking may be beneficial (protein block followed by peroxidase quenching)

  • Species-specific serum matching the secondary antibody host is recommended

  • Extended blocking (2+ hours) may improve signal-to-noise ratio

• Antibody dilution optimization:

  • Systematically test dilution series beyond manufacturer recommendations

  • For HRP-conjugated antibodies, higher dilutions often reduce background without compromising specific signal

• Washing modifications:

  • Increase washing duration and volume

  • Consider adding 0.05-0.1% Tween-20 to wash buffers

  • For tissues with high lipid content (where PEX11A is abundant), mild detergents in wash buffers can reduce non-specific binding

How should researchers design experiments to study PEX11A in relation to peroxisomal disorders?

When investigating PEX11A in peroxisomal dysfunction:

• Model selection: Consider both in vitro cell models and in vivo animal models with relevance to peroxisomal disorders. PEX11A deficiency has been linked to impaired peroxisome elongation and reduced fatty acid oxidation capacity .

• Experimental controls:

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

  • Negative controls: Consider PEX11A knockdown/knockout models

  • Technical controls: Include secondary-only controls for HRP systems to assess non-specific binding

• Measurement parameters:

  • Beyond protein expression, assess functional peroxisome parameters

  • Consider correlating PEX11A levels with peroxisome number, size, and distribution

  • Evaluate downstream metabolic effects such as changes in fatty acid oxidation

• Time-course considerations:

  • Acute vs. chronic models may show different PEX11A regulation patterns

  • Developmental timing may be critical, especially in neural differentiation models

What quantification approaches provide reliable analysis of PEX11A expression patterns?

For accurate quantification of PEX11A:

• Western blot quantification:

  • Normalize to appropriate loading controls (membrane protein controls may be more suitable than cytosolic proteins)

  • Use densitometry with linear range validation

  • Consider biological replicates (n≥3) for statistical validity

• Immunohistochemistry quantification:

  • For HRP-based detection, DAB intensity can be quantified with calibrated image analysis

  • Consider H-score methods that account for both staining intensity and percentage of positive cells

  • For subcellular localization studies, co-localization coefficients with peroxisomal markers provide valuable data

• Flow cytometry applications:

  • For cell populations, flow cytometry with permeabilization protocols optimized for membrane proteins can provide quantitative data

  • Dual labeling with peroxisomal markers can identify population heterogeneity

How can researchers effectively study interactions between PEX11A and coatomer proteins?

Given PEX11A's role in mediating binding of coatomer proteins to the peroxisomal membrane , interaction studies require specialized approaches:

• Co-immunoprecipitation strategies:

  • Use membrane-compatible lysis buffers that preserve protein-protein interactions

  • Consider crosslinking approaches for transient interactions

  • Sequential immunoprecipitation may provide cleaner results for complex formation analysis

• Proximity ligation assays:

  • These can provide spatial resolution of interactions in intact cells

  • Particularly valuable for distinguishing peroxisomal membrane interactions from other cellular compartments

• FRET/BRET approaches:

  • For live-cell dynamics, energy transfer techniques can reveal interaction kinetics

  • Requires careful control experiments to account for membrane protein constraints

• Visualization strategies:

  • Super-resolution microscopy combined with appropriate antibody labeling can reveal colocalization at the nanoscale level

  • For HRP-conjugated antibodies in electron microscopy, DAB polymerization provides excellent spatial resolution of peroxisomal membranes

What emerging techniques show promise for advancing PEX11A research?

Several cutting-edge approaches hold potential for deepening our understanding of PEX11A biology:

• CRISPR-based approaches:

  • Endogenous tagging of PEX11A for live-cell imaging

  • Precise genome editing to study specific domains and post-translational modifications

  • CRISPRi/CRISPRa for temporal control of expression levels

• Proximity labeling techniques:

  • BioID or APEX2 fusions with PEX11A to identify novel interaction partners

  • Particularly valuable for understanding membrane protein complexes

• Single-cell analyses:

  • Examining cell-to-cell variability in PEX11A expression and localization

  • Correlating with metabolic parameters at single-cell resolution

• Advanced imaging:

  • Live-cell super-resolution imaging to track peroxisome dynamics

  • Correlative light and electron microscopy for structural-functional relationships