PRPH Antibody

What is Peripherin (PRPH) and why is it an important target for antibody-based detection in neurological research?

Peripherin is a 53.7 kDa (typically observed at 57 kDa) type III intermediate filament protein encoded by the PRPH gene. It is predominantly expressed in peripheral nervous system (PNS) neurons and select central nervous system (CNS) neurons, including some cortical neurons, hippocampal neurons, and ventral horn spinal cord motor neurons . Peripherin plays crucial roles in:

• Maintaining structural integrity of neuronal cytoskeleton

• Supporting axonal development and elongation

• Facilitating axonal regeneration after injury

• Contributing to neuronal responses under stress conditions

As a neuronal marker, peripherin antibodies are particularly valuable for identifying and studying PNS neurons, certain CNS subpopulations, and for investigating neuronal structural maintenance and regeneration mechanisms .

What are the key applications for Peripherin antibodies in neuroscience research?

Peripherin antibodies demonstrate utility across multiple experimental applications:

Researchers should note that peripherin antibodies have been extensively validated in human, mouse, and rat samples, with particularly robust detection in spinal cord and peripheral nerve tissues .

How should researchers select between polyclonal and monoclonal Peripherin antibodies?

The choice between polyclonal and monoclonal antibodies depends on experimental needs:

Polyclonal Peripherin Antibodies:

• Recognize multiple epitopes on the peripherin protein

• Provide enhanced sensitivity for detecting low-abundance targets

• Useful for applications like Western blotting and immunoprecipitation

• Available in rabbit and chicken hosts (e.g., chicken polyclonal antibodies show excellent specificity at dilutions of 1:5,000-1:10,000 for WB)

Monoclonal Peripherin Antibodies:

• Target specific epitopes with high consistency

• Minimize batch-to-batch variation

• Preferred for longitudinal studies requiring consistent results

• Example: Mouse monoclonal clone 7C5 is highly specific for the ~57 kDa peripherin protein and performs well on aldehyde-fixed tissues

For experiments requiring high reproducibility across multiple studies, monoclonal antibodies are recommended. For maximum detection sensitivity, polyclonal antibodies may be preferable .

What control samples should be used when validating Peripherin antibody performance?

Appropriate positive and negative controls are essential when validating peripherin antibody performance:

Positive Controls:

• Rat or mouse spinal cord homogenate (consistently high peripherin expression)

• Peripheral nerve tissue (dorsal root ganglia, sciatic nerve)

• PC-12 cells (rat pheochromocytoma cell line with reliable peripherin expression)

• COLO 320 cells (human colorectal cancer cells expressing peripherin)

Negative Controls:

• Non-neuronal tissues (e.g., liver, kidney)

• Primary antibody omission controls

• Blocking peptide controls where available

• IgG isotype controls matching the host species of the primary antibody

Researchers should observe a specific band at approximately 57 kDa in Western blots of positive control samples, with no significant bands in negative controls .

How can researchers optimize immunostaining protocols for peripherin detection in different neural tissues?

Optimizing peripherin immunostaining requires tissue-specific considerations:

For Paraffin-Embedded CNS Tissues:

• Perform antigen retrieval using TE buffer (pH 9.0) for optimal epitope exposure

• Alternative: Citrate buffer (pH 6.0) if TE buffer yields insufficient signal

• Block with 5-10% normal serum from the secondary antibody host species

• Incubate with primary antibody at 1:50-1:500 dilution (overnight at 4°C)

• Wash extensively (3-5× with PBS-0.1% Tween 20)

• Apply fluorophore-conjugated secondary antibody (1:500-1:1000)

For PNS Tissues and Frozen Sections:

• Fix briefly (10-15 minutes) with 4% paraformaldehyde

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

• Block with serum-containing buffer (1-2 hours)

• Incubate with antibody at higher dilution (1:200-1:1000)

• Consider tyramide signal amplification for detecting low abundance targets

For challenging tissues or when background is problematic, titrate antibody concentration carefully and extend washing steps. Both monoclonal (e.g., clone 7C5) and polyclonal antibodies perform well in immunohistochemistry, though monoclonal antibodies may provide cleaner background in some tissues .

What are the key considerations when using Peripherin antibodies to study neurodegenerative diseases like ALS?

Peripherin has significant implications in amyotrophic lateral sclerosis (ALS) research, requiring specific experimental considerations:

Technical Considerations:

• Use high-sensitivity detection methods (e.g., enhanced chemiluminescence for WB)

• Include positive controls from spinal cord tissues

• Examine multiple regions (cortex, spinal cord, motor end plates)

• Consider dual-labeling with TDP-43 or SOD1 antibodies

Analytical Approaches:

• Quantify peripherin-positive aggregates in motor neurons

• Assess peripherin expression changes relative to disease progression

• Compare peripherin isoform ratios between healthy and diseased tissues

• Evaluate peripherin phosphorylation status using phospho-specific antibodies

Researchers should note that peripherin undergoes upregulation during periods of trophic stress and that mutations in the peripherin gene have been associated with ALS. This makes peripherin a valuable diagnostic marker for the ballooned axons seen in ALS .

How can researchers identify and troubleshoot potential cross-reactivity issues with Peripherin antibodies?

Cross-reactivity concerns with peripherin antibodies can be addressed through systematic validation:

Potential Cross-Reactivity Sources:

• Other intermediate filament proteins (particularly type III filaments like vimentin, GFAP, and desmin)

• Alternatively spliced peripherin isoforms

• Post-translationally modified forms of peripherin

Validation Methods:

• Western Blot Analysis:

  • Compare banding patterns across multiple tissues

  • Look for expected 57 kDa band (primary peripherin species)

  • Check for absence of bands in non-neuronal tissues

• Immunodepletion Studies:

  • Pre-absorb antibody with recombinant peripherin protein

  • Confirm elimination of signal in positive control samples

• Knockout Validation:

  • Test antibody in PRPH knockout models or PRPH-knockdown cell lines

  • Confirm absence of signal compared to wild-type controls

• Dual Labeling:

  • Co-stain with antibodies against other intermediate filament proteins

  • Confirm appropriate co-localization patterns and absence of unexpected overlap

To minimize cross-reactivity, researchers can use monoclonal antibodies targeting unique peripherin epitopes or highly purified polyclonal antibodies that have undergone affinity purification against the immunizing antigen .

What methodological approaches are recommended for studying peripherin's role in axonal transport and neurite extension?

To investigate peripherin's functions in axonal transport and neurite extension:

Cell Culture Models:

• Establish primary sensory neuron cultures from dorsal root ganglia

• Use PC-12 cells (can be differentiated with NGF to form neurites)

• Consider compartmentalized culture systems (e.g., microfluidic chambers) to isolate axons

Molecular Manipulation Techniques:

• Perform PRPH gene knockdown using siRNA or shRNA

• Overexpress wild-type or mutant peripherin constructs

• Use CRISPR-Cas9 for precise gene editing of peripherin

Imaging Approaches:

• Live-cell imaging of fluorescently tagged peripherin to track dynamics

• Super-resolution microscopy for detailed cytoskeletal organization

• Proximity ligation assays to identify protein interaction partners

Functional Assays:

• Measure neurite outgrowth rates following peripherin manipulation

• Assess axonal transport using fluorescent cargo (e.g., labeled mitochondria)

• Quantify growth cone dynamics in response to peripherin modulation

These approaches can elucidate peripherin's role in axon elongation, regeneration after injury, and its inhibitory effect on neurite extension in specific neuronal populations like type II spiral ganglion neurons .

How does peripherin interact with other neuronal intermediate filament proteins, and what methods can detect these interactions?

Peripherin can form heteropolymers with other neuronal intermediate filaments, requiring specialized detection methods:

Known Interaction Partners:

• Neurofilament light chain (NEFL)

• Neurofilament medium chain (NEFM)

• Neurofilament heavy chain (NEFH)

• α-internexin (INA)

Detection Methods:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate peripherin and probe for associated proteins

  • Use gentle lysis conditions to preserve cytoskeletal interactions

  • Consider crosslinking to stabilize transient interactions

• Proximity Ligation Assay (PLA):

  • Visualize protein-protein interactions in situ with <40nm resolution

  • Enables quantification of interaction frequency in different cellular compartments

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag peripherin and potential partners with appropriate fluorophore pairs

  • Measure energy transfer as indication of molecular proximity

• Double Immunogold Electron Microscopy:

  • Use differentially sized gold particles to label peripherin and partners

  • Visualize ultrastructural co-localization at nanometer resolution

Research has shown that assembly of neuronal intermediate filaments, including peripherin networks, may be regulated by RAB7A. The formation of these filamentous networks is critical for maintaining structural integrity under mechanical stress .

What are the current methodologies for studying peripherin gene mutations and their relationship to neurological disorders?

Research into peripherin mutations and neurological disorders employs diverse methodological approaches:

Genetic Analysis Techniques:

• Next-Generation Sequencing:

  • Screen PRPH exons in patient cohorts with neurological disorders

  • Identify rare variants and assess their population frequency

• Digital Droplet PCR:

  • Quantify peripherin transcript levels with high precision

  • Detect subtle expression changes in disease states

• RNA-Seq Analysis:

  • Profile peripherin isoform expression patterns

  • Identify disease-specific alterations in splicing

Functional Characterization Methods:

• In Vitro Systems:

  • Express mutant peripherin in cell models

  • Assess impact on filament formation, stability, and cellular localization

  • Evaluate interactions with other cytoskeletal components

• Animal Models:

  • Generate transgenic mice expressing human peripherin mutations

  • Analyze motor function, neurodegeneration, and peripherin aggregation

  • Perform longitudinal studies to correlate with human disease progression

• Patient-Derived iPSCs:

  • Differentiate into motor neurons or sensory neurons

  • Compare cytoskeletal organization between normal and mutant cells

  • Test potential therapeutic interventions in disease-relevant cell types

These approaches have established links between peripherin mutations and ALS, where peripherin-positive protein aggregates are frequently observed in affected motor neurons. The upregulation of peripherin after nerve injury also suggests its role in neuronal stress responses .