PEX1 Antibody, HRP conjugated

What is PEX1 and why is it significant in research?

PEX1 (peroxisomal biogenesis factor 1) is an ATPase that facilitates the recycling of the peroxisome matrix protein receptor PEX5. It plays a critical role in generating and maintaining peroxisomes in eukaryotic cells. PEX1 is particularly significant in research because it is the most commonly affected peroxin in human peroxisome biogenesis disorders (PBDs) . With a calculated molecular weight of 143 kDa (1283 amino acids), PEX1 works within the peroxisomal exportomer complex to enable proper peroxisome function, including metabolism of very long chain fatty acids, detoxification of reactive oxygen species, and biosynthesis of plasmalogens contained in myelin .

What applications are PEX1 antibodies typically used for?

PEX1 antibodies are utilized across multiple experimental techniques in peroxisome research. The most common applications include:

These applications allow researchers to detect PEX1 expression, localization, and interaction with other peroxisomal proteins in various tissue and cell types . Published research demonstrates successful use of PEX1 antibodies in knockout/knockdown experiments, Western blotting, immunohistochemistry, and immunofluorescence studies across human and mouse samples .

What cell and tissue types show reliable PEX1 expression for antibody testing?

Based on validation data, PEX1 antibody demonstrates positive Western blot detection in Jurkat and HeLa cells. For immunohistochemistry, human liver cancer tissue and human kidney tissue show reliable PEX1 expression. Immunofluorescence applications have successfully detected PEX1 in HepG2, A431, and HeLa cells . When designing experiments, these validated cell types provide reliable positive controls for antibody performance assessment.

What is the optimal protocol for conjugating PEX1 antibody with HRP?

The optimal protocol for HRP conjugation to PEX1 antibody follows these methodological steps:

• Ensure the purified antibody is in 10-50mM amine-free buffer (MES, MOPS, HEPES, PBS) with pH 6.5-8.5 .

• Add the appropriate Modifier reagent to the antibody sample (1μl of Modifier per 10μl of antibody) .

• Transfer the antibody-Modifier mixture directly onto lyophilized HRP Mix and gently resuspend.

• Incubate at room temperature (20-25°C) for 3 hours in dark conditions.

• Add Quencher reagent (1μl per 10μl of antibody) and mix gently.

• Allow 30 minutes for quenching to complete before use .

This methodology produces ready-to-use HRP-conjugated PEX1 antibody without requiring additional purification steps, though the conjugation efficacy should be validated for each specific experimental application.

What is the optimal antibody-to-HRP ratio for maximizing sensitivity while maintaining specificity?

The antibody-to-HRP ratio significantly impacts conjugate performance. Based on established protocols, two primary ratios are recommended:

The 1:4 ratio (antibody:HRP) provides optimal sensitivity for most applications, while the 1:1 ratio may be preferable when working with samples containing potentially cross-reactive proteins . Researchers should conduct titration experiments to determine the optimal ratio for their specific experimental system, as the ideal ratio may vary based on the antibody's affinity and the target protein's abundance.

How should HRP-conjugated PEX1 antibody be stored to maintain activity?

For optimal stability and retention of activity, HRP-conjugated PEX1 antibody should be stored at -20°C in a buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the conjugated antibody remains stable for one year after preparation. Aliquoting is not necessary for -20°C storage of standard preparations, though it may be beneficial for frequently used conjugates to prevent freeze-thaw cycles . For conjugates prepared using 20μl sizes, note that they contain 0.1% BSA as a stabilizer .

How can PEX1 antibody be used to investigate peroxisome biogenesis disorders?

PEX1 antibody serves as a critical tool for investigating peroxisome biogenesis disorders (PBDs) through multiple methodological approaches:

• Differential diagnosis: Using immunofluorescence with PEX1 antibody can reveal abnormal localization patterns of peroxisomal proteins. In cells with functional PEX1, PTS1-containing proteins show punctate distribution co-localizing with peroxisome membrane proteins like ABCD3. In contrast, cells with PEX1 defects show cytosolic localization of these proteins .

• Mutation analysis: Comparing PEX1 protein levels between wild-type and mutant cells (particularly the common G843D mutation) using Western blot analysis can quantify the extent of protein instability. Research shows the G843D mutation significantly reduces PEX1 protein levels due to enhanced proteasomal degradation .

• Therapeutic evaluation: PEX1 antibody can assess the efficacy of gene therapy approaches by measuring restored PEX1 expression and function. For example, IHC using tagged PEX1 antibodies has confirmed transgene expression in retinal tissues following AAV-mediated gene delivery .

What methodological approaches can differentiate between mutant and wild-type PEX1 in research models?

Researchers can employ several methodological approaches to distinguish mutant from wild-type PEX1:

• Co-immunoprecipitation with mass spectrometry: This technique reveals differential binding partners between wild-type and mutant PEX1. Studies show PEX1G843D exhibits significantly reduced association with PEX6 and PEX26, confirming assembly defects in the hexameric complex .

• Epitope tagging combined with immunofluorescence: Using N- or C-terminal FLAG-tagged PEX1 constructs allows visualization of protein localization and expression. This approach has demonstrated that mutant PEX1G843D shows altered cellular distribution compared to wild-type .

• CRISPR-Cas9 gene editing with restriction enzyme validation: Creating precise mutations (e.g., G843D) alongside silent mutations that introduce restriction enzyme sites enables rapid screening of edited clones via PCR amplification and restriction digestion .

• Rescue experiments with proteasome inhibitors: Treating PEX1 mutant cells with proteasome inhibitors can restore protein levels, confirming degradation as the primary mechanism for reduced abundance rather than transcriptional changes .

How can immunofluorescence with PEX1 antibody reveal peroxisome assembly defects?

Immunofluorescence using PEX1 antibody, particularly in combination with markers for peroxisomal proteins, provides valuable insights into peroxisome assembly defects through these methodological steps:

• Co-staining cells with PEX1 antibody and antibodies against PTS1-containing matrix proteins and membrane proteins like ABCD3.

• In normal cells, PTS1-containing proteins show punctate localization co-localizing with ABCD3, indicating proper peroxisome matrix protein import .

• In cells with PEX1 mutations, PTS1 proteins appear diffusely cytosolic due to import failure, while PEX5 remains abnormally accumulated at peroxisome membranes .

• Following interventions (gene therapy, drug treatments), restoration of punctate PTS1 protein localization and cytosolic PEX5 distribution indicates functional recovery of the PEX1-dependent import mechanism .

This approach has proven valuable in assessing recovery of peroxisome function in both murine models and human cell lines with PEX1 mutations .

What are common sources of non-specific binding with HRP-conjugated PEX1 antibody and how can they be mitigated?

Non-specific binding with HRP-conjugated PEX1 antibody can arise from several sources, each requiring specific mitigation strategies:

• Inappropriate antibody-to-HRP ratio: Excessive HRP conjugation can increase non-specific interactions. Solution: Optimize conjugation ratios, testing both 1:4 and 1:1 antibody:HRP ratios to determine the most specific signal for your application .

• Buffer incompatibilities: Pre-conjugation buffer components (particularly primary amines, carriers, and preservatives) can interfere with conjugation efficiency. Solution: Ensure antibody is in 10-50mM amine-free buffer (pH 6.5-8.5) before conjugation and remove interfering components through dialysis if necessary .

• Cross-reactivity with endogenous peroxidases: Particularly problematic in tissues with high peroxidase activity. Solution: Include adequate peroxidase quenching steps (e.g., 0.3% H₂O₂ in methanol) in your IHC protocol .

• Inadequate blocking: Insufficient blocking leads to increased background. Solution: Optimize blocking conditions using BSA, normal serum, or commercial blocking reagents specific to your sample type .

How can antigen retrieval methods be optimized for PEX1 detection in different tissue types?

Optimizing antigen retrieval for PEX1 detection requires tissue-specific approaches:

• For liver and kidney tissues: The recommended primary method is heat-induced epitope retrieval (HIER) with TE buffer at pH 9.0. This has been validated for human liver cancer and kidney tissues .

• Alternative approach: When TE buffer pH 9.0 provides suboptimal results, citrate buffer (pH 6.0) can serve as an effective alternative, particularly for samples with high fixation levels .

• Methodology optimization:

  • Begin with the validated retrieval method (TE buffer pH 9.0)

  • If signal is weak, increase retrieval time in increments of 5 minutes

  • For excessive background, reduce retrieval time or switch to citrate buffer

  • For each new tissue type, compare both buffer systems side-by-side

  • Document optimal conditions for future reference

• Tissue-specific considerations: Different tissue types may require unique retrieval conditions based on protein expression levels, fixation methods, and tissue architecture .

How can researchers validate the functionality of HRP-conjugated PEX1 antibody in new experimental systems?

Validating HRP-conjugated PEX1 antibody functionality in new experimental systems requires a systematic approach:

• Positive control testing: Begin with validated cell lines known to express PEX1 (Jurkat, HeLa, HepG2, A431) to establish baseline performance metrics .

• Knockout/knockdown validation: Utilize PEX1-null cell lines (like engineered HepG2 cells) or siRNA-mediated knockdown to confirm signal specificity. The signal should be absent or significantly reduced in these systems .

• Multi-technique confirmation: Validate antibody performance across multiple techniques (WB, IF, IHC) to ensure consistent target recognition across different protein conformations .

• Titration experiments: Perform dilution series experiments to determine optimal antibody concentration for each specific application and cell/tissue type .

• Epitope-tagged controls: Compare antibody performance against epitope-tagged versions of PEX1 (e.g., HA-tagged or FLAG-tagged PEX1) to confirm specific target recognition .

• Functional recovery assay: In PEX1-deficient systems, measure restoration of peroxisome function following genetic complementation to confirm the antibody detects functionally relevant protein .

How is PEX1 antibody being used in gene therapy research for peroxisome biogenesis disorders?

PEX1 antibody plays a crucial role in evaluating gene therapy approaches for peroxisome biogenesis disorders through several methodological applications:

• Vector expression verification: Researchers use PEX1 antibody to confirm successful expression of therapeutic transgenes. For instance, in AAV-mediated gene therapy for ZSD (Zellweger spectrum disorder), immunohistochemistry with anti-HA antibodies detected human PEX1-HA expression in retinal tissues following subretinal injection .

• Quantitative expression analysis: Western blotting with PEX1 antibody allows researchers to measure transgene expression levels relative to endogenous protein, confirming sufficient expression for therapeutic effect. Studies demonstrate markedly increased PEX1 protein levels in tissues receiving HsPEX1 gene therapy .

• Functional recovery assessment: Using immunofluorescence co-localization studies, researchers can assess whether transgene expression restores proper peroxisomal protein localization. In mouse models with PEX1-G844D mutation (equivalent to human G843D), PEX1 antibody helps confirm that gene therapy can recover proper PTS1 protein import and PEX5 recycling .

• Long-term expression monitoring: PEX1 antibody enables tracking of therapeutic protein expression over time, critical for understanding the durability of gene therapy approaches .

What insights does research provide about the effect of PEX1 mutations on protein stability and function?

Recent research using PEX1 antibody has revealed critical insights about mutation effects on protein stability and function:

• G843D mutation mechanism: The common G843D mutation (G844D in mice) impairs PEX1's assembly with PEX6, as demonstrated through immunoprecipitation and mass spectrometry. This assembly defect promotes proteasomal degradation of the mutant protein, resulting in reduced steady-state levels .

• Complex assembly dynamics: Immunoprecipitation studies show significantly reduced association of PEX1G843D with partners PEX6 and PEX26, suggesting the mutation disrupts formation of the functional hexameric AAA+ ATPase complex required for peroxisome protein import .

• Degradation pathway: Research using proteasome inhibitors demonstrates that PEX1G843D undergoes enhanced ubiquitin-mediated proteasomal degradation. Fusion of a deubiquitinase to PEX1G843D significantly hinders this degradation in mammalian cells, suggesting potential therapeutic approaches .

• Functional threshold concept: Overexpression studies reveal that PEX1G843D can restore peroxisome function if protein levels exceed a critical threshold, indicating that stabilization strategies may provide therapeutic benefit without needing to correct the underlying mutation .

How can researchers measure the impact of therapeutic interventions on PEX1 expression and function?

Researchers can employ several methodological approaches using PEX1 antibody to assess therapeutic interventions:

• Quantitative Western blotting: This technique measures changes in PEX1 protein levels following treatment. Studies show proteasome inhibitors can restore PEX1G843D protein levels, demonstrating potential for protein stabilization approaches .

• Peroxisomal import assay: Immunofluorescence co-localization of PTS1-containing proteins with peroxisome membrane markers serves as a functional readout of PEX1 activity. This approach has demonstrated that gene therapy can restore proper peroxisomal protein localization in mouse models .

• PEX5 recycling assessment: Visualizing PEX5 localization provides insight into PEX1 function, as PEX1 facilitates PEX5 recycling. In wild-type cells, PEX5 is primarily cytosolic, while in PEX1-deficient cells, it appears punctate and co-localizes with peroxisome membranes .

• Proteasomal degradation analysis: Cycloheximide chase experiments combined with Western blotting can measure PEX1 protein half-life changes in response to treatments, providing insight into protein stabilization efficacy .

• Physiological outcome measures: Beyond direct protein measurements, researchers assess improvements in tissue-specific functions, such as visual function in retinal gene therapy models, to correlate protein-level changes with functional recovery .