PEX11A Antibody, Biotin conjugated

What is PEX11A and what is its functional role in peroxisome biology?

PEX11A (Peroxisomal Biogenesis Factor 11 alpha) is a peroxisomal membrane protein of approximately 28 kDa that plays a crucial role in peroxisomal proliferation. PEX11A functions to:

• Promote membrane protrusion and elongation on the peroxisomal surface

• Regulate peroxisome division through direct mechanisms rather than indirect metabolic effects

• Potentially mediate binding of coatomer proteins to the peroxisomal membrane

Research using genetic knockout models demonstrates that PEX11 proteins are unique in their ability to promote peroxisome division across multiple species. Importantly, PEX11 overexpression experiments conclusively show that it promotes peroxisome division even in the absence of peroxisomal metabolic activity, indicating that PEX11A affects organelle abundance directly rather than through metabolic pathways .

What are the optimal experimental conditions for using biotin-conjugated PEX11A antibodies in immunological assays?

When working with biotin-conjugated PEX11A antibodies, researchers should optimize several parameters:

Dilution Optimization:

• For ELISA applications: Begin with 1:500-1:2,000 dilution ranges and perform titration experiments to determine optimal signal-to-noise ratios

• Optimal dilutions/concentrations should be determined empirically by each investigator as performance may vary between antibody lots

Buffer Conditions:

• Standard buffers contain: 0.01M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol

• Note that Proclin is a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only

Storage and Stability:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze/thaw cycles to maintain antibody functionality

• If aliquoting is necessary, prepare single-use volumes to prevent degradation

Control Experiments:

• Include appropriate isotype controls: Rabbit IgG is the appropriate control for polyclonal PEX11A antibodies

• Validate antibody specificity using PEX11A knockout or knockdown cells when possible

How can researchers distinguish between the three human PEX11 isoforms (α, β, γ) in experimental systems?

Distinguishing between PEX11 isoforms requires careful attention to antibody selection and validation protocols:

Sequence Homology Considerations:

• The three human PEX11 isoforms (PEX11α, PEX11β, and PEX11γ) share conserved domains but have distinct N-terminal regions

• Multiple sequence alignment analysis reveals that the N-terminal PEX19 binding site (BS1) is highly conserved among species but varies between isoforms

Antibody Selection Strategy:

• Choose antibodies raised against non-conserved regions to ensure specificity

• The antibody described in the search results targets amino acids 106-219 of human PEX11A , a region that contains unique sequences

Validation Methods:

• Perform Western blot analysis with recombinant proteins of all three isoforms to confirm specificity

• Validate with genetic models: use cells overexpressing or lacking specific PEX11 isoforms

• Consider yeast two-hybrid analysis for interaction studies, as demonstrated for the binding of human PEX11β N-terminal fragment with full-length human PEX19

Functional Differentiation:
Different PEX11 isoforms show distinct functional properties that can be used for identification:

• PEX11α is particularly responsive to peroxisome proliferators

• PEX11β knockout has more severe phenotypes than PEX11α knockout in mice

• PEX11γ has not shown clear interaction with PEX19 in some experimental systems

What are the critical considerations for immunohistochemical application of PEX11A antibodies?

When planning immunohistochemistry experiments with PEX11A antibodies, researchers should consider:

Tissue Preparation and Fixation:

• Formalin-fixed, paraffin-embedded (FFPE) tissues require appropriate antigen retrieval methods

• For PEX11A detection, citrate buffer (pH 6.0) heat-induced epitope retrieval is often effective

• Optimize fixation time as overfixation may mask epitopes

Antibody Validation Controls:

• Positive controls: Include tissues known to express PEX11A (liver, kidney proximal tubules)

• Negative controls: Use tissues from PEX11A knockout models or omit primary antibody

• Peptide competition assays can confirm specificity

Detection Systems:

• For biotin-conjugated antibodies, use streptavidin-based detection systems

• Be aware of potential endogenous biotin interference in certain tissues (liver, kidney)

• Consider avidin/biotin blocking steps for tissues with high endogenous biotin

Co-localization Studies:

• Pair with established peroxisomal markers (catalase, PEX14) for confirmation of peroxisomal localization

• Use confocal microscopy for precise co-localization analysis

• Quantify peroxisome abundance by counting peroxisomes per cell section (reference range: ~230 ± 52 peroxisomes per section in wild-type cells)

How are PEX11A antibodies utilized to investigate peroxisome proliferation mechanisms?

PEX11A antibodies serve as powerful tools for investigating peroxisome proliferation through several methodological approaches:

Overexpression Studies:

• Track peroxisome morphological changes following PEX11A overexpression

• Use PEX11A antibodies to confirm expression levels by Western blot

• Combine with fluorescent peroxisomal markers (e.g., DsRed-SKL) to visualize peroxisome abundance changes

Kinetic Analysis:

• Monitor temporal changes in peroxisome morphology and abundance

• Previous studies have identified three kinetically distinct steps in PEX11β-induced peroxisome proliferation observable within 1.5-2 hours after expression

Mutagenesis Approaches:

• Generate PEX11A mutants lacking specific domains or binding sites

• Use antibodies to confirm expression and localization of mutant proteins

• Key mutations to consider include those affecting PEX19 binding sites (e.g., L35P mutation in the conserved N-terminal region)

Knockout/Knockdown Experiments:

• Compare peroxisome abundance in wild-type vs. PEX11A-deficient cells

• Quantitative analysis shows PEX11β−/− cells have approximately half the peroxisome abundance compared to wild-type cells (128 ± 32 vs. 230 ± 52 peroxisomes per section)

• Use antibodies to confirm knockout/knockdown efficiency

What is the relationship between PEX11A deficiency and disease pathophysiology?

Research has revealed important connections between PEX11A and disease states:

Hypertension and Kidney Disease:

• Pex11a deficiency impairs the abundance of functional peroxisomes in proximal tubule cells

• This deficiency aggravates tubulointerstitial damage and hypertension

• Peroxisomes play a critical role in the pathological process of chronic kidney disease

Metabolic Pathway Disruptions:

• PEX11β−/− mice show partial deficiency in two distinct peroxisomal metabolic pathways:

  • Ether lipid synthesis

  • Very long chain fatty acid oxidation

• These deficiencies likely result from indirect effects on peroxisome membrane structure or dynamics

Experimental Approaches:

• Use PEX11A antibodies to quantify protein levels in disease models

• Employ tissue-specific knockout models to elucidate organ-specific roles

• Combine with metabolomic analysis to identify affected pathways

Therapeutic Implications:

• Pex11a and the peroxisome system represent potential novel therapeutic targets for prevention of hypertensive chronic kidney disease

• Screening compounds that enhance PEX11A expression or function may have therapeutic value

How can the PEX19 binding sites in PEX11A be experimentally characterized?

The interaction between PEX19 and PEX11A is critical for peroxisomal targeting and can be investigated through several methodologies:

Peptide Array Analysis:

• Synthesize overlapping 15-mer peptides with 2-amino acid shifts covering the PEX11A sequence

• Probe with purified GST-PEX19 fusion proteins

• Immunodetect binding using anti-GST antibodies

• Consider binding positive when at least three consecutive peptide spots show interaction

Binding Site Mapping:
Research has identified three potential PEX19 binding sites (BS) in Trypanosoma brucei PEX11:

• BS1: amino acids 13-35 (N-terminal, cytosolic)

• BS2: amino acids 77-99 (near first transmembrane domain)

• BS3: amino acids 139-159 (near second transmembrane domain)

Mutagenesis Studies:

• Generate PEX11A deletion mutants lacking specific binding sites

• Create point mutations in conserved residues (e.g., L35P mutation)

• Assess effects on:

  • Protein stability (steady-state concentration)

  • Subcellular localization

  • Direct interaction with PEX19

Yeast Two-Hybrid Analysis:

• Use as validation method for identified binding sites

• Results from studies with human PEX11 family members:

How can researchers optimize Western blot protocols for PEX11A detection?

Western blot optimization for PEX11A detection requires attention to several technical parameters:

Sample Preparation:

• For membrane proteins like PEX11A, use appropriate extraction buffers containing mild detergents

• Consider specialized membrane protein extraction kits

• Load approximately 25μg protein per lane for optimal detection

Antibody Dilutions:

• Primary antibody (anti-PEX11A): 1:500-1:2,000

• Secondary antibody: 1:10,000 dilution (HRP-conjugated anti-rabbit IgG for rabbit polyclonal antibodies)

Blocking Conditions:

• Use 3% non-fat dry milk in TBST for optimal blocking

• Alternative blocking agents: 5% BSA may provide better results for phospho-specific antibodies

Detection System:

• For biotin-conjugated antibodies, use streptavidin-HRP conjugates

• Standard HRP-conjugated secondary antibodies work for unconjugated primary antibodies

• ECL-based detection systems provide good sensitivity for PEX11A detection

• Recommended exposure time: approximately 60 seconds

Expected Results:

• Predicted molecular weight: 28 kDa

• Validate specificity by comparing multiple cell lines

• Consider using PEX11A knockout/knockdown samples as negative controls

What are the experimental approaches to resolve contradictory findings about PEX11A's direct versus indirect role in peroxisome metabolism?

Historical controversy exists regarding whether PEX11 proteins directly regulate metabolism or primarily affect peroxisome division. Several experimental approaches can address this question:

Metabolic Activity in PEX11-Overexpressing Cells:

• Express PEX11 in cells lacking functional peroxisomal β-oxidation pathway

• Compare peroxisome abundance in these cells versus controls

• Studies show that PEX11 overexpression promotes peroxisome division even in the absence of peroxisomal metabolic activity (e.g., in pox1 mutant yeast lacking acyl-CoA oxidase)

Analysis of Multiple Metabolic Pathways:

• Examine various peroxisomal metabolic functions in PEX11-deficient models

• PEX11β knockout mice show deficiencies in multiple unrelated pathways (ether lipid synthesis and very long chain fatty acid oxidation)

• This suggests an indirect effect rather than direct regulation of specific enzymes

Peroxisome Abundance in Metabolite-Free Conditions:

• Culture PEX11β+/+ and PEX11β−/− cells in serum-free medium (lacking peroxisomal metabolic substrates)

• Quantify peroxisome abundance by immunofluorescence

• PEX11β−/− cells show reduced peroxisome abundance (~50%) even without metabolic substrates

Timing of Events:

• Establish temporal relationship between PEX11 expression, peroxisome division, and metabolic changes

• Use inducible expression systems to control timing of PEX11 expression

• Monitor metabolic and morphological parameters with time-course experiments

What role does the N-terminal PEX19 binding site play in PEX11 function across species?

The N-terminal PEX19 binding site of PEX11 shows evolutionary conservation and functional significance:

Conservation Analysis:

• Multiple sequence alignment reveals conservation of the N-terminal PEX19 binding site (BS1) among diverse species including parasites, yeast, humans, and plants

• This conservation suggests fundamental importance to PEX11 function

Functional Studies:

• In Trypanosoma brucei: Deletion of BS1 reduces steady-state protein levels but some PEX11 still targets to glycosomes

• In yeast: The L35P mutation in the conserved binding site abolishes interaction with PEX19 and causes protein instability and mislocalization to mitochondria

• In humans: Of the three PEX11 isoforms, only PEX11β shows clear interaction with PEX19 through its N-terminal region

Localization Studies:

• Wild-type PEX11-GFP targets to peroxisomes (co-localizes with DsRed-SKL peroxisomal marker)

• PEX11-GFP with L35P mutation partially mislocalizes to tubular structures identified as mitochondria (confirmed by MitoTracker staining)

• Immunoblot analysis shows dramatically reduced steady-state levels of the L35P mutant protein

Mechanistic Model:

• The PEX19 binding site serves dual functions:

  • Stabilization of PEX11 protein

  • Proper targeting to peroxisomes

• In the absence of proper PEX19 binding, PMPs including PEX11 may be mislocalized to other membranes (particularly mitochondria) or degraded