PEX19-1 Antibody

What is PEX19 and why is it important in cellular research?

PEX19 is a peroxisomal biogenesis factor essential for early peroxisomal development. It serves dual functions: as a cytosolic chaperone that stabilizes newly synthesized peroxisomal membrane proteins (PMPs) by binding to their hydrophobic membrane-spanning domains, and as an import receptor that targets these PMPs to the peroxisome membrane by interacting with the integral membrane protein PEX3 . Its critical role in peroxisome biogenesis makes it a valuable target for studying organelle biogenesis, protein trafficking, and related disorders.

Which applications are PEX19 antibodies most commonly used for?

PEX19 antibodies have demonstrated utility across multiple experimental techniques, including:

• Western blotting (WB): Typically at dilutions of 1:1000-1:4000 for polyclonal antibodies and 1:5000-1:50000 for monoclonal antibodies

• Immunohistochemistry (IHC): At dilutions of 1:50-1:500

• Immunofluorescence/Immunocytochemistry (IF/ICC): At dilutions of 1:200-1:800

• Immunoprecipitation (IP): Using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• ELISA applications

What is the difference between polyclonal and monoclonal PEX19 antibodies?

Polyclonal PEX19 antibodies (e.g., ab95959, 14713-1-AP) are typically produced in rabbits and recognize multiple epitopes of the PEX19 protein, offering high sensitivity but potentially variable specificity between lots . Monoclonal PEX19 antibodies (e.g., 68555-1-Ig) are produced from a single B-cell clone, providing consistent reproducibility and high specificity for a single epitope . Polyclonal antibodies may be preferable for detection of low-abundance PEX19, while monoclonal antibodies offer better specificity for distinguishing between PEX19 and closely related proteins.

What specimen types can be analyzed using PEX19 antibodies?

Commercial PEX19 antibodies have been validated for:

• Human samples: Widely tested across multiple cell lines including Jurkat, K-562, HeLa, HEK-293, A549, and LNCaP cells

• Mouse samples: Particularly liver tissue and heart tissue

• Rat samples: Primarily liver tissue

• Pig samples: Liver tissue

Other species may show reactivity due to sequence homology, but validation would be required.

What are the optimal fixation and antigen retrieval methods for PEX19 immunostaining?

For immunohistochemistry applications with PEX19 antibodies, the following protocol has shown optimal results:

• Fixation: 10% neutral buffered formalin

• Antigen retrieval: TE buffer (pH 9.0) is recommended as the primary method

• Alternative method: Citrate buffer (pH 6.0) can be used as an alternative

For immunofluorescence, a brief (10-15 minute) fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.3% Triton X-100 has been successfully employed in experimental studies .

How can I validate the specificity of my PEX19 antibody?

Multiple validation approaches should be employed:

• Perform western blot analysis and confirm the appropriate molecular weight (calculated: 33 kDa; observed: 35-40 kDa)

• Include appropriate positive controls (e.g., Jurkat cells, K-562 cells, or liver tissue)

• Include a negative control (PEX19-deficient cells if available, or cells treated with PEX19 siRNA)

• Consider using PEX19 knockout/knockdown cells as a critical control to confirm specificity

• Check for cross-reactivity with recombinant PEX19 protein

• Perform peptide competition assays to confirm epitope specificity

What controls should be included when studying PEX19 in peroxisome biogenesis experiments?

Rigorous experimental design should include:

• Positive controls: Normal human fibroblasts expressing PEX19 (e.g., GM5756-T cell line)

• Negative controls: PEX19-deficient human fibroblasts (e.g., PBD399 cell line)

• Additional control: PEX3-deficient human fibroblasts (e.g., PBD400-TI)

• Functional controls: Complementation with wild-type PEX19 should restore peroxisomal function in PEX19-deficient cells

• Control for farnesylation: Use PEX19/C296A mutant (non-farnesylated form) to study the role of farnesylation in PEX19 function

How can I investigate PEX19 interaction with peroxisomal membrane proteins?

Multiple complementary approaches have been validated:

• Co-immunoprecipitation assays:

  • Express epitope-tagged PMPs with PEX19 or 3xHA-PEX19

  • Lyse cells in hypotonic buffer and discard membrane fractions

  • Immunoprecipitate with anti-HA antibodies

  • Immunoblot with antibodies specific to the PMPs of interest

• Pulse-chase experiments for interaction dynamics:

  • Express PMP34/13xmyc in cells with or without PEX19

  • Pulse label with [35S]methionine for 1 hour

  • Chase with excess cold methionine for varying times

  • Immunoprecipitate at each time point

  • Determine half-life through autoradiography and immunoblotting

• Blot overlay technique:

  • Express and purify recombinant PEX19

  • Separate by SDS-PAGE, transfer to membranes and renature

  • Probe with 35S-labeled human integral PMPs

  • For quantitative analysis, use labeled 6xHis-PEX14 and apply at various concentrations

• Nuclear mislocalization assay:

  • Generate NLS-PEX19 fusion construct

  • Co-express with PMPs of interest

  • Analyze subcellular localization of PMPs by immunofluorescence

  • PEX19 interaction is indicated by nuclear accumulation of PMPs

What methods can determine the subcellular distribution of PEX19?

Two complementary approaches provide insight into PEX19 localization:

• Differential centrifugation:

  • Homogenize cells in hypotonic buffer containing protease inhibitors

  • Separate cytosolic and membrane fractions through centrifugation

  • Analyze fractions by immunoblotting with anti-PEX19 antibodies

  • Include marker proteins for quality control (e.g., catalase for peroxisomes)

• Differential permeabilization and release assay:

  • Incubate intact cells with varying concentrations of digitonin

  • Collect released material at each concentration

  • Assay for PEX19, cytosolic marker (LDH), and peroxisomal marker (catalase)

  • Compare release profiles to determine compartmentalization

How can I analyze the role of PEX19 farnesylation in its function?

The farnesyl modification of PEX19 at cysteine-296 can be studied through:

• Structural analysis:

  • NMR-derived structure shows the farnesyl moiety is buried in an internal hydrophobic cavity

  • Chemical shift analysis can reveal conformational changes upon farnesylation

• Functional analysis:

  • Generate PEX19/C296A mutant (non-farnesylated form)

  • Compare wild-type and mutant PEX19 in functional complementation assays

  • Assess ability to rescue peroxisome biogenesis in PEX19-deficient cells

• Binding studies with farnesylation mutants:

  • Generate point mutations that affect farnesyl recognition (e.g., M255R)

  • Assess chemical shift differences using NMR

  • Compare PMP binding affinity between wild-type and mutant forms

What should I consider if my PEX19 antibody shows unexpected molecular weight?

While the calculated molecular weight of PEX19 is 33 kDa, it is typically observed at 35-40 kDa on SDS-PAGE . This discrepancy may be due to:

• Post-translational modifications, particularly farnesylation

• Structural features that affect electrophoretic mobility

• Different isoforms or splice variants

• Protein degradation during sample preparation

If you observe significantly different molecular weights, consider:

• Verifying antibody specificity with positive controls

• Using freshly prepared samples with protease inhibitors

• Trying different sample preparation conditions

• Running a recombinant PEX19 standard alongside your samples

How can I differentiate between class 1 and class 2 mPTS-containing proteins in PEX19 studies?

Research has identified two classes of membrane PTS (mPTS) with different PEX19 dependencies:

Class 1 mPTS proteins:

• Directly bound by PEX19

• Import is PEX19-dependent

• Can be identified through PEX19 binding assays (co-IP, blot overlay)

• Stabilized by PEX19 in the cytosol

Class 2 mPTS proteins:

• Not bound by PEX19

• Import is PEX19-independent

• Cannot be recovered in PEX19 immunoprecipitates

• Not stabilized by PEX19 expression

For experimental distinction:

• Perform binding assays with purified PEX19 and your protein of interest

• Compare protein stability in PEX19-expressing versus PEX19-deficient cells

• Assess localization in cells treated with PEX19-specific siRNA

What factors might influence the detection of PEX19-PMP interactions in experimental settings?

Several factors can affect the detection of PEX19-PMP interactions:

• Expression levels: Overexpression can lead to non-physiological interactions

• Membrane isolation: PEX19-PMP interactions may be transient and disrupted during membrane preparation

• Detergent conditions: Critical for maintaining protein-protein interactions

• Farnesylation status: The farnesyl group is important for PEX19 function

• Assay sensitivity: Different methods have varying sensitivities for detecting interactions

• PMP topology: The presentation of binding sites may be affected by experimental conditions

• Competing proteins: Other cellular proteins may compete for binding

To optimize detection, consider:

• Using multiple complementary approaches (co-IP, blot overlay, nuclear mislocalization)

• Careful titration of expression levels

• Including appropriate controls for specificity

• Using mild detergent conditions or detergent-free methods when possible

How should I interpret varying results across different PEX19 antibodies?

Differences between antibodies may reflect:

• Epitope location: Different antibodies recognize different regions of PEX19

• Affinity differences: Varying binding strengths affect detection sensitivity

• Cross-reactivity: Some antibodies may recognize related proteins

• Post-translational modifications: Modifications may mask certain epitopes

• Isoform specificity: Some antibodies may preferentially detect specific isoforms

For reliable interpretation:

• Use multiple antibodies targeting different epitopes

• Include positive and negative controls

• Consider the specific application requirements (native vs. denatured protein)

• Verify key findings with alternative detection methods

How can PEX19 antibodies be used to study peroxisome biogenesis disorders?

PEX19 antibodies serve as valuable tools for investigating peroxisome biogenesis disorders (PBDs):

• Diagnostic applications:

  • Immunoblotting to assess PEX19 protein levels in patient-derived cells

  • Immunofluorescence to evaluate peroxisome abundance and morphology

  • Combined with PEX3 analysis to distinguish different PBD complementation groups

• Functional complementation studies:

  • Transfect PBD patient fibroblasts with wild-type or mutant PEX19

  • Use immunofluorescence with peroxisomal marker antibodies (e.g., anti-catalase) to assess rescue

  • Calculate relative rescue activity using established protocols

• PMP stability assessment:

  • Compare PMP levels in normal versus PBD patient fibroblasts

  • Pulse-chase experiments to determine PMP half-lives

  • Immunofluorescence to assess PMP mislocalization (often to mitochondria)

What methodologies can evaluate the chaperone function of PEX19?

The chaperone activity of PEX19 can be assessed through:

• In vivo stability assays:

  • Express PMPs in PEX19-expressing versus PEX19-deficient cells

  • Compare protein abundance through immunoblotting

  • Pulse-chase experiments to determine protein half-lives

• Binding to newly-synthesized PMPs:

  • Co-express PEX19 and PMPs in peroxisome-deficient cells

  • Perform sequential immunoprecipitation to isolate PEX19-PMP complexes

  • Assess binding kinetics through time-course experiments

• Assessment of PMP aggregation:

  • Monitor PMP solubility in the presence or absence of PEX19

  • Compare detergent-soluble versus insoluble fractions

  • Examine mislocalization to mitochondria in PEX19-deficient cells

• Analysis of specific chaperone domains:

  • Generate PEX19 mutants with alterations in putative chaperone regions

  • Test their ability to bind and stabilize PMPs

  • Particularly relevant for PEX11 studies, which contain only one PEX19-binding site far from its transmembrane domains