PEX26 Antibody

What is PEX26 and why is it important in cellular research?

PEX26 functions as a peroxisomal docking factor that anchors PEX1 and PEX6 to peroxisome membranes. It is therefore required for the formation of the PEX1-PEX6 AAA ATPase complex, which mediates the extraction of the PEX5 receptor from peroxisomal membranes . PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins, highlighting its essential role in peroxisome biogenesis . The significance of PEX26 extends to clinical research, as its dysfunction results in peroxisome biogenesis disorders (PBDs) leading to Zellweger spectrum disorders (ZSDs) .

What types of PEX26 antibodies are available for research purposes?

PEX26 antibodies are predominantly available as rabbit polyclonal antibodies that recognize human PEX26. These antibodies are typically generated using recombinant fragments or fusion proteins containing segments of the human PEX26 protein (often within the N-terminal 1-200 amino acid region) . The canonical human PEX26 protein has a reported length of 305 amino acid residues and a mass of 33.9 kDa, with observed molecular weights in experimental conditions typically ranging from 30-34 kDa .

What applications are PEX26 antibodies validated for?

Most commercially available PEX26 antibodies have been validated for:

PEX26 antibodies have been successfully used to detect endogenous PEX26 in human cell lines including K562, A431, and HEK-293 cells .

How can I optimize Western blot protocols for PEX26 detection?

For optimal Western blot detection of PEX26:

• Sample preparation: Use whole cell lysates from cells with known PEX26 expression (K562, A431, or HEK-293 cells are good positive controls) .

• Protein loading: Load 20-30 μg of total protein per lane.

• Gel percentage: Use 10-12% polyacrylamide gels for optimal separation of proteins in the 30-34 kDa range.

• Transfer conditions: Transfer to PVDF or nitrocellulose membranes using standard protocols (wet transfer often yields better results for membrane proteins).

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute PEX26 antibody 1:1000-1:4000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence for detection.

• Expected band: Look for a specific band at approximately 30-34 kDa .

What are the recommended controls when studying PEX26 knockdown or knockout effects?

When designing experiments involving PEX26 silencing or knockout:

• Positive controls: Include cell lines with confirmed high expression of PEX26 (such as K562 cells) .

• Negative controls:

  • For siRNA experiments: Use scrambled siRNA sequences (sgSCR) .

  • For CRISPR experiments: Use single guide scrambled (sgSCR) controls .

• Validation approach: Confirm knockdown/knockout efficiency using:

  • Western blot to assess protein reduction

  • qRT-PCR to confirm reduction in mRNA levels

  • Immunofluorescence to visualize changes in subcellular localization

• Functional validation: Assess peroxisomal function by measuring:

  • Peroxisomal matrix protein import using PTS1/PTS2 reporter constructs

  • Levels of very long chain fatty acids (VLCFAs) which accumulate in PEX26 dysfunction

  • Pexophagy rates, which increase upon PEX26 silencing

How can PEX26 be visualized in its native peroxisomal context?

For visualization of PEX26 in peroxisomes:

• Co-localization studies: Use PEX26 antibodies in combination with established peroxisomal markers:

  • Catalase (peroxisomal matrix protein)

  • PTS1-containing proteins (using anti-PTS1 antibodies)

  • PEX14 (peroxisomal membrane protein)

• Immunofluorescence protocol:

  • Fix cells in 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1-0.2% Triton X-100 (5-10 minutes)

  • Block with 2-5% BSA or normal serum

  • Incubate with PEX26 antibody (typically 1:100-1:500 dilution)

  • Use appropriate fluorophore-conjugated secondary antibodies

  • Counter-stain for peroxisomal markers

• Super-resolution microscopy: For detailed analysis of PEX26 localization and interaction with other peroxins, consider techniques such as STED or STORM microscopy.

How can PEX26 antibodies be used to study the mechanism of pexophagy in disease models?

PEX26 silencing has been shown to enhance pexophagy and sensitize drug-resistant cancer cells to therapy . To investigate pexophagy mechanisms:

• Autophagosome isolation:

  • Perform autophagic vesicle enrichment from control and experimental conditions

  • Use PEX26 antibodies in combination with autophagy markers (LC3-II) for co-immunoprecipitation or Western blot analysis

  • Mass spectrometry on autophagosome-enriched fractions can reveal changes in peroxisomal protein content

• Imaging-based approaches:

  • Transfect cells with fluorescently tagged peroxisomal markers

  • Track co-localization with autophagy markers (LC3, LAMP1) in response to PEX26 modulation

  • Use live-cell imaging to monitor pexophagy dynamics

• Biochemical validation:

  • Measure changes in peroxisomal enzyme activities (e.g., catalase, ACOX1)

  • Assess peroxisome number and morphology using PEX26 antibodies in combination with other peroxisomal markers

  • Quantify lipid peroxidation products as indicators of peroxisomal stress

The experimental design should include ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase) inhibition as a control, as this has been shown to rescue the effects of PEX26 silencing on pexophagy .

What approaches can be used to study the PEX26-PEX19-PEX3 interaction complex?

The PEX19-dependent peroxisomal targeting of PEX26 involves complex formation with PEX19 in the cytosol and subsequent interaction with PEX3 at the peroxisomal membrane . To investigate this complex:

• In vitro binding assays:

  • Use recombinant proteins to study direct interactions

  • Co-immunoprecipitation with PEX26 antibodies to pull down interacting partners

  • Perform GST pulldown assays with tagged proteins

• Domain mapping:

  • Generate truncated constructs to identify interaction domains

  • Focus on the N-terminal half of PEX26 (aa 29-174) for PEX6 binding

  • Analyze the C-terminal transmembrane domain and positively charged residues in the C segment for PEX19 binding

• Proximity-based labeling:

  • Use BioID or APEX2 fused to PEX26 to identify proximal proteins in living cells

  • Confirm interactions using PEX26 antibodies in combination with antibodies against putative interaction partners

• Functional reconstitution:

  • Utilize semi-intact cell systems to study the import of purified PEX19-PEX26 complexes

  • Test the requirement for PEX3 by using PEX3-depleted cells or PEX3 mutants (e.g., PEX3-W104A, which fails to recruit PEX26)

How can PEX26 antibodies be used to investigate alternative splicing of PEX26 in pathological conditions?

PEX26 undergoes alternative splicing to produce several splice forms, including PEX26-Δex5 (lacking exon 5) . To study these variants:

• Splice variant-specific detection:

  • Design epitope mapping experiments to determine if existing PEX26 antibodies detect all splice variants

  • Consider generating splice variant-specific antibodies if needed

• Expression analysis:

  • Use RT-PCR with splice junction-spanning primers to amplify specific variants

  • Confirm protein expression of variants using Western blot with PEX26 antibodies

  • Correlate variant expression with disease progression or severity

• Functional characterization:

  • Express individual splice variants in PEX26-deficient cells to assess rescue of peroxisomal defects

  • Use PEX26 antibodies to confirm expression and localization of variants

  • Analyze interaction profiles of different splice variants with peroxins like PEX1 and PEX6

Why might PEX26 antibodies show unexpected molecular weight bands in Western blots?

Researchers may encounter unexpected bands when using PEX26 antibodies in Western blot applications. Potential explanations include:

• Alternative splice variants: PEX26 undergoes alternative splicing, producing variants like PEX26-Δex5 and PEX26-Δex2 , which would appear at different molecular weights.

• Post-translational modifications: Check if the unexpected bands could represent phosphorylated, ubiquitinated, or otherwise modified forms of PEX26.

• Cross-reactivity: Some antibodies may cross-react with structurally similar proteins. Validate specificity using PEX26 knockdown or knockout controls.

• Protein degradation: PEX26 may undergo proteolytic processing during sample preparation. Use fresh samples and appropriate protease inhibitors.

• Sample preparation conditions: Membrane proteins can form aggregates that are resistant to complete denaturation. Try varying the detergent concentration or heating conditions.

How can I determine if my PEX26 antibody is suitable for studying peroxisome biogenesis disorders?

To validate PEX26 antibodies for studying peroxisome biogenesis disorders:

• Patient cell lines testing:

  • Test the antibody on cell lines from patients with confirmed PEX26 mutations

  • Compare with control cell lines to establish detection sensitivity

• Variant detection:

  • Determine if the antibody can detect known pathogenic variants of PEX26

  • Note that some mutations may affect the epitope recognized by the antibody

• Functional correlation:

  • Assess correlation between PEX26 protein levels (detected by the antibody) and biochemical markers of peroxisomal function

  • Measure very long chain fatty acid (VLCFA) levels, which are elevated in peroxisomal disorders

• Immunofluorescence validation:

  • Confirm that the antibody can detect differences in PEX26 localization between normal and patient cells

  • Look for mislocalization or reduced peroxisomal staining in patient samples

What are the key considerations when designing experiments to study PEX26's role in cancer therapy resistance?

Low expression levels of PEX26 have been associated with prolonged patient survival in lymphoma, lung cancer, and melanoma cohorts . When investigating PEX26 in cancer therapy resistance:

• Experimental models:

  • Use paired sensitive/resistant cancer cell lines

  • Consider isogenic models where PEX26 is the only variable

  • Include xenograft models to validate in vitro findings

• Silencing approaches:

  • Compare transient (siRNA) vs. stable (shRNA, CRISPRi) suppression of PEX26

  • Use inducible systems to control the timing of PEX26 suppression

• Therapeutic combinations:

  • Test PEX26 suppression in combination with standard therapies (e.g., histone deacetylase inhibitors for lymphoma)

  • Monitor drug sensitivity using appropriate cytotoxicity or apoptosis assays

• Mechanistic investigations:

  • Monitor peroxisome numbers and function using PEX26 antibodies

  • Assess changes in redox status, as peroxisomes play key roles in cellular redox balance

  • Investigate altered lipid metabolism as a potential mechanism of therapy resistance

• Clinical correlation:

  • Use PEX26 antibodies for immunohistochemical analysis of patient samples

  • Correlate PEX26 expression with treatment response and survival outcomes