PEX11G Antibody

What is PEX11G and why is it important in peroxisome research?

PEX11G (peroxisomal biogenesis factor 11 gamma, also known as PEX11C) is a 241 amino acid multi-pass membrane protein with a molecular weight of approximately 27 kDa that belongs to the peroxin-11 family . It is localized to peroxisomes and promotes membrane protrusion and elongation on the peroxisomal surface . PEX11G is crucial in studies of peroxisome biogenesis, membrane dynamics, and organelle proliferation. The PEX11 family proteins are the only factors known to promote peroxisome division in multiple species, making them essential for understanding peroxisomal dynamics and function .

How do I select the appropriate PEX11G antibody for my experiment?

Selection should be based on:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, ELISA)

• Species reactivity: Confirm reactivity with your species of interest (human, mouse, rat, etc.)

• Epitope location: Consider whether N-terminal, C-terminal, or internal epitopes are more suitable for your research question

• Validation data: Review published literature and manufacturer validation data

For example, the 15744-1-AP antibody has been validated for WB, IF, IHC, and ELISA applications with demonstrated reactivity to human and mouse samples .

What are the optimal working dilutions for different applications of PEX11G antibodies?

Always titrate the antibody in your specific testing system to obtain optimal results .

What is the recommended protocol for detecting PEX11G in Western blot applications?

For optimal Western blot detection of PEX11G:

• Sample preparation: Prepare whole cell lysates from appropriate cells (e.g., PC-12 cells, human/mouse testis tissue have shown positive results)

• Protein loading: Load 10-20 μg of protein per lane

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal separation

• Transfer: Transfer to PVDF or nitrocellulose membranes

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

• Primary antibody: Dilute PEX11G antibody (e.g., 1:500-1:3000) in blocking buffer and incubate overnight at 4°C

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (e.g., 1:1000-1:5000)

• Detection: Use ECL detection system

• Expected band: Look for a band at approximately 27 kDa

How should I perform immunohistochemistry with PEX11G antibodies?

For IHC detection of PEX11G:

• Tissue preparation: Use paraffin-embedded tissue sections (4-6 μm thickness)

• Antigen retrieval: Perform antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with serum

• Primary antibody: Apply diluted PEX11G antibody (1:20-1:200) and incubate overnight at 4°C

• Secondary antibody and detection: Use appropriate detection system compatible with your primary antibody

• Counterstaining: Counterstain with hematoxylin

• Positive controls: Human kidney tissue has shown positive reactivity

What controls should I include when performing experiments with PEX11G antibodies?

Why might I observe multiple bands in Western blot when using PEX11G antibodies?

Multiple bands could result from:

• Isoforms: PEX11G exists as at least two isoforms due to alternative splicing

• Post-translational modifications: Modifications like phosphorylation can alter migration patterns

• Degradation products: Sample preparation issues can cause protein degradation

• Cross-reactivity: Antibody may cross-react with other PEX11 family members (PEX11α, PEX11β)

• Non-specific binding: Insufficient blocking or high antibody concentration

To troubleshoot:

• Optimize sample preparation to minimize degradation

• Adjust blocking conditions and antibody dilutions

• Perform peptide competition assays to verify specificity

• Compare results with different PEX11G antibodies targeting different epitopes

How can I improve weak or absent signal when detecting PEX11G?

To enhance PEX11G detection:

• Antibody concentration: Increase primary antibody concentration (within recommended range)

• Incubation time: Extend primary antibody incubation to overnight at 4°C

• Antigen retrieval: Optimize antigen retrieval method (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Protein loading: Increase protein amount (especially for low-abundance samples)

• Sensitive detection: Use more sensitive detection systems (ECL Plus, SuperSignal West Femto)

• Fresh antibody: Ensure antibody hasn't degraded due to improper storage or handling

• Expression verification: Confirm PEX11G expression in your sample type

How can I distinguish between PEX11G and other PEX11 family proteins (PEX11α, PEX11β) in my studies?

To specifically detect PEX11G among other PEX11 family members:

• Epitope selection: Choose antibodies targeting unique regions of PEX11G

  • The glycine-rich region (amino acids 159-182) is present in PEX11β but absent in PEX11α and PEX11γ

  • C-terminal sequences differ between PEX11 isoforms

• Validation methods:

  • Perform Western blot analysis with recombinant PEX11α, PEX11β, and PEX11γ proteins

  • Use cells with knockout/knockdown of specific PEX11 isoforms

  • Compare antibody reactivity patterns with known expression profiles

• Complementary approaches:

  • Use RT-PCR to confirm isoform-specific mRNA expression

  • Employ fluorescent protein tags for specific isoform visualization

How can I use PEX11G antibodies to study peroxisome division mechanisms?

To investigate peroxisome division using PEX11G antibodies:

• Co-localization studies:

  • Perform immunofluorescence co-staining of PEX11G with other peroxisomal markers (PEX14, catalase)

  • Examine co-localization with fission machinery components (DRP1, FIS1, MFF)

• Functional analysis:

  • Compare peroxisome morphology in wild-type vs. PEX11G knockdown/knockout cells

  • Assess PEX11G localization during peroxisome proliferation stimulated by treatments (e.g., fibrates)

• Protein interactions:

  • Use co-immunoprecipitation with PEX11G antibodies to identify interaction partners

  • Perform proximity ligation assays to detect in situ protein interactions

• Dynamic studies:

  • Use time-lapse imaging with immunofluorescence to track peroxisome division events

  • Compare localization patterns during different cell cycle stages

Research has shown that PEX11 proteins promote peroxisome division independently of peroxisome metabolism and may regulate division by recruiting the dynamin-related GTPase DNM1L to the peroxisomal membrane .

How can I resolve contradictory findings when studying PEX11G function using antibody-based approaches?

When facing contradictory results:

• Antibody validation comparison:

  • Verify specificity of antibodies used in contradictory studies

  • Test multiple antibodies targeting different epitopes of PEX11G

  • Perform knockout/knockdown controls for each antibody

• Experimental conditions analysis:

  • Compare cell types, culture conditions, and treatments

  • Assess peroxisome induction status (basal vs. proliferation-induced)

  • Consider effects of cell cycle, differentiation state, or stress conditions

• Methodological differences:

  • Evaluate fixation methods (critical for peroxisome morphology preservation)

  • Compare sample preparation protocols

  • Assess detection sensitivity differences

• Biological context:

  • Consider functional redundancy between PEX11 family members

  • Evaluate species-specific differences in PEX11G function

  • Assess tissue-specific expression patterns and functions

Research shows that PEX11β can compensate for loss of PEX11G in some contexts, and that different PEX11 proteins may have specialized functions in different tissues or conditions .

How can PEX11G antibodies be used to study peroxisome dysfunction in disease models?

PEX11G antibodies can facilitate disease research through:

• Expression analysis:

  • Compare PEX11G expression levels in normal vs. diseased tissues

  • Assess correlation between PEX11G levels and disease progression

  • Examine tissue-specific alterations in peroxisome abundance

• Morphological studies:

  • Analyze peroxisome size, number, and distribution in patient samples

  • Correlate peroxisome morphology changes with disease phenotypes

  • Assess effects of therapeutic interventions on peroxisome dynamics

• Functional investigations:

  • Investigate relationship between PEX11G expression and peroxisomal metabolic functions

  • Study interaction between PEX11G and disease-related proteins

  • Evaluate PEX11G post-translational modifications in disease states

• Model systems:

  • Generate and characterize PEX11G knockout/knockdown disease models

  • Study compensatory mechanisms between PEX11 family members

  • Test therapeutic approaches targeting peroxisome division

Research has shown connections between peroxisome dysfunction and neurodegenerative disorders, with a novel defect in peroxisome division linked to a homozygous non-sense mutation in the PEX11β gene .

What methodological approaches can distinguish the specific roles of PEX11G versus PEX11α and PEX11β in experimental systems?

To dissect specific roles of PEX11 isoforms:

• Sequential knockout analysis:

  • Generate single, double, and triple knockout models of PEX11 isoforms

  • Assess peroxisome morphology, abundance, and function in each model

  • Identify isoform-specific and redundant functions

• Domain swapping experiments:

  • Generate chimeric constructs exchanging domains between PEX11 isoforms

  • Express PEX11G with the C-terminal tail of PEX11α or PEX11β

  • Identify functional domains responsible for specific activities

• Tissue-specific analysis:

  • Compare expression patterns of PEX11 isoforms across tissues

  • Generate tissue-specific knockout models

  • Assess peroxisome parameters in different tissues

• Protein interaction mapping:

  • Identify unique and shared interaction partners for each isoform

  • Perform targeted mutagenesis of interaction domains

  • Correlate interaction profiles with functional differences

Research has revealed that PEX11β, but not PEX11α, can restore peroxisome division in PEX11β-deficient cells, and that the C-terminal region of PEX11β is essential for this function .

How can I apply phosphorylation-specific antibodies to study PEX11G regulation?

To investigate PEX11G phosphorylation:

• Phospho-specific antibody development:

  • Identify potential phosphorylation sites in PEX11G

  • Generate phospho-specific antibodies against these sites

  • Validate antibody specificity with phosphatase treatments

• Functional studies:

  • Compare phosphorylated vs. total PEX11G during peroxisome proliferation

  • Generate phospho-mimetic and phospho-deficient PEX11G mutants

  • Correlate phosphorylation status with peroxisome morphology and division

• Regulatory mechanisms:

  • Identify kinases and phosphatases that regulate PEX11G

  • Study signaling pathways that control PEX11G phosphorylation

  • Investigate cross-talk between different post-translational modifications

Recent research has demonstrated phosphorylation-dependent activation of peroxisome proliferator-activated receptor, suggesting similar mechanisms may regulate PEX11G function .

What are the recommended approaches for studying PEX11G interactions with membrane remodeling machinery?

To investigate PEX11G's role in membrane remodeling:

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with PEX11G antibodies

  • Use proximity ligation assays to detect in situ interactions

  • Apply FRET/BRET techniques to study dynamic interactions

• Membrane dynamics analysis:

  • Use super-resolution microscopy to visualize membrane deformation

  • Apply correlative light and electron microscopy (CLEM)

  • Employ live-cell imaging with fluorescently tagged PEX11G

• Reconstitution systems:

  • Generate artificial membrane systems with purified PEX11G

  • Study membrane curvature induction in vitro

  • Assess interactions with other membrane-remodeling proteins

• Structure-function analysis:

  • Identify membrane-binding domains in PEX11G

  • Generate and test domain-specific mutants

  • Correlate structural features with membrane remodeling capacity