RPH3A Antibody

What is RPH3A and what cellular functions does it perform?

RPH3A (Rabphilin 3A) is a neuronal C2 domain tandem containing protein with multiple critical functions in the nervous system. It plays essential roles in:

• Docking and fusion steps of regulated exocytosis

• Recruitment to synaptic vesicle membranes by RAB3A in a GTP-dependent manner

• Modulation of synaptic vesicle trafficking and calcium-triggered neurotransmitter release

• Formation of a ternary complex with GluN2A and PSD-95 to regulate NMDA receptor stability in postsynaptic compartments

• Exocytosis of arginine vasopressin hormone

RPH3A is primarily expressed in the brain, with dense distribution in synaptic regions, including the rat brain, retina, neuromuscular junctions, and dendritic spines of the lateral structural domain of the postsynaptic density (PSD) .

What is the molecular structure of RPH3A and its functional domains?

RPH3A contains three main structural domains with distinct functions:

The N-terminal domain is responsible for binding with GluN2A, as demonstrated by co-localization assays with truncated mutants . The carboxy-terminal C2 domains bind calcium ions and phosphatidylinositol 4,5-bisphosphate containing lipid vesicles .

What techniques are commonly used to detect RPH3A in research samples?

RPH3A can be detected using multiple experimental approaches:

For immunodetection methods, antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 is often recommended for optimal results .

How should RPH3A antibodies be stored and handled for optimal results?

For optimal performance of RPH3A antibodies:

• Store at -20°C in aliquots to avoid freeze-thaw cycles

• Most commercial antibodies are stable for one year after shipment when stored properly

• Common storage buffers include PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• For diluted working solutions, use blocking buffers containing 3-5% BSA or similar proteins

• Keep on ice when working with the antibody and return to storage promptly after use

What controls should be included when using RPH3A antibodies?

Proper experimental controls are essential:

• Positive controls: Rat or mouse brain tissue extracts, SH-SY5Y cells, or COLO 320 cells are commonly used

• Negative controls: Samples lacking primary antibody or tissues known not to express RPH3A

• Specificity controls: Use of blocking peptides (when available) to confirm antibody specificity

• For phospho-specific RPH3A antibodies, treatment with phosphatases can serve as negative controls

• siRNA or CRISPR-mediated knockdown of RPH3A can provide additional validation

How can anti-RPH3A antibodies be used as diagnostic markers for neuroendocrine disorders?

Anti-rabphilin-3A antibodies have emerged as important diagnostic markers for certain neuroendocrine disorders:

• Lymphocytic infundibuloneurohypophysitis (LINH): Anti-rabphilin-3A antibodies showed 100% sensitivity (4/4 patients) and 100% specificity in distinguishing LINH from other sellar/suprasellar masses that are clinically difficult to differentiate .

• Central Diabetes Insipidus (CDI): In a study of 15 consecutive CDI patients, anti-rabphilin-3A antibodies were found in:

  • 4 of 5 LINH cases (80%)

  • 3 of 4 lymphocytic panhypophysitis (LPH) cases (75%)

  • 1 of 2 sarcoidosis cases (50%)

  • 1 intracranial germinoma case

This makes anti-rabphilin-3A antibodies valuable for differentiating CDI etiologies, particularly compared to anti-vasopressin-cell antibodies which lack specificity .

Detection methodology involves Western blotting using recombinant human rabphilin-3A protein lysate as the antigen and patient serum as the primary antibody. A protein band at 76 kDa appearing in cells transfected with rabphilin-3A protein (but not in control cells) is considered positive .

What is the significance of RPH3A phosphorylation and how can phospho-specific antibodies aid in its investigation?

RPH3A phosphorylation, particularly at Ser237, plays a crucial role in regulating its functions:

• Phospho-specific antibodies targeting Ser237 allow researchers to investigate the dynamic regulation of RPH3A activity in response to cellular stimuli

• These antibodies can be used in multiple applications including Western blot, immunohistochemistry, and ELISA

• Phosphorylation state can be correlated with specific neuronal functions or pathological conditions

When designing experiments with phospho-specific RPH3A antibodies:

• Include appropriate controls (phosphatase-treated samples)

• Consider time-course experiments to capture dynamic phosphorylation changes

• Pair with total RPH3A antibodies to calculate the phosphorylation ratio

• Use kinase inhibitors to identify regulatory pathways

How does RPH3A interact with GluN2A and PSD-95 to regulate NMDA receptor stability?

RPH3A forms a ternary complex with GluN2A (an NMDA receptor subunit) and PSD-95 (a postsynaptic scaffold protein) to regulate NMDA receptor stability at synapses:

• The N-terminal Rab-binding domain of RPH3A is responsible for interaction with GluN2A, as demonstrated through co-localization assays with truncated mutants

• Disruption of either GluN2A/RPH3A or PSD-95/RPH3A interactions leads to decreased stability of the GluN2A/PSD-95 complex

• This destabilization induces a rapid decrease in GluN2A-containing NMDARs at the postsynaptic membrane within 10-15 minutes

• Electron microscopy studies show RPH3A localization at postsynaptic membranes near PSDs, strategically positioned to prevent receptor endocytosis

• Blocking endocytosis with dynamin-inhibitor dynasore prevents GluN2A reduction at the cell surface induced by disrupting the GluN2A/RPH3A complex

To investigate these interactions, researchers can use:

• Co-immunoprecipitation assays to detect protein-protein interactions

• Time-lapse confocal imaging with SEP-GluN2A to track receptor dynamics

• Electrophysiological measurements of NMDAR currents

• Peptide interference techniques to disrupt specific protein interactions

What role does RPH3A play in cerebral ischemia-reperfusion injury and how can it be studied?

RPH3A shows dynamic changes following cerebral ischemia-reperfusion injury:

• mRNA and protein levels of RPH3A significantly increase in the brain penumbra in rat MCAO/R (Middle Cerebral Artery Occlusion/Reperfusion) models

• RPH3A is primarily distributed in neurons and astrocytes, with expression significantly increased after MCAO/R

• Downregulation of RPH3A worsens cerebral infarct, neuronal death, and behavioral, cognitive, and memory impairments in rats after MCAO/R

• In vitro studies show ischemia-reperfusion upregulates RPH3A protein levels and secretion in astrocytes but decreases RPH3A protein levels in neurons

Experimental approaches to study RPH3A in cerebral ischemia include:

• Time-course analysis: Measuring RPH3A protein and mRNA levels at 6, 12, 24, 72, 120 hours, and 1 week after MCAO/R

• Cell-type specific analysis: Using immunofluorescence co-staining with neuronal (NeuN), astrocyte (GFAP), and microglial (Iba1) markers

• Loss-of-function studies: Lentiviral delivery of RPH3A-RNAi to specific brain regions using stereotaxic surgery

• In vitro models: Oxygen-glucose deprivation and reoxygenation in primary neuronal and astrocytic cultures

What are the optimal protocols for validating RPH3A antibody specificity?

Comprehensive validation of RPH3A antibodies ensures reliable results:

Advanced validation approaches include:

• RNAseq correlation for enhanced validation

• Mass spectrometry confirmation of immunoprecipitated protein

• Immunohistochemistry on tissue arrays with known RPH3A expression patterns

How do different fixation and sample preparation methods affect RPH3A antibody performance?

Sample preparation significantly impacts RPH3A antibody performance:

• Fixation: 4% paraformaldehyde is commonly used for immunofluorescence studies , but may affect epitope accessibility

• Antigen retrieval: TE buffer (pH 9.0) or citrate buffer (pH 6.0) are recommended for IHC applications to expose masked epitopes

• Permeabilization: 0.1% Triton X-100 is effective for accessing intracellular epitopes in immunofluorescence

• Blocking: 3-5% BSA in PBS is typically used to reduce non-specific binding

• Primary antibody incubation: Overnight incubation at 4°C generally yields optimal results for RPH3A detection

Different applications may require specific optimization:

• For Western blot: RIPA lysis buffer with protease inhibitors is recommended

• For immunofluorescence: Longer primary antibody incubation (3 hours at room temperature or overnight at 4°C)

• For electron microscopy: Special fixation protocols may be necessary to preserve ultrastructural details

How can I design experiments to study the differentially regulated expression of RPH3A in neurons versus astrocytes?

To investigate cell-type specific RPH3A regulation:

• Dual immunofluorescence labeling:

  • Co-stain with RPH3A antibody and cell-type specific markers (NeuN for neurons, GFAP for astrocytes)

  • Quantify relative fluorescence intensity using ImageJ or similar software

• Cell isolation and Western blot:

  • Use magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) to isolate neurons and astrocytes

  • Perform Western blot analysis on isolated populations to compare RPH3A expression levels

• In vitro models:

  • Culture primary neurons and astrocytes separately

  • Apply identical experimental conditions (e.g., oxygen-glucose deprivation)

  • Compare changes in RPH3A expression and secretion

• Single-cell RNA sequencing:

  • Analyze RPH3A transcript levels in different cell populations

  • Correlate with protein expression data from immunohistochemistry

Key experimental considerations include using identical antibody concentrations across samples, blinded quantification of results, and appropriate statistical analysis to account for cell-type specific differences.

What are common issues when using RPH3A antibodies and how can they be resolved?

How can I optimize RPH3A antibody dilutions for different applications?

Optimal dilutions vary by application and specific antibody:

Remember that optimal dilutions may vary between tissue types, fixation methods, and detection systems. Document successful conditions for reproducibility.

How can RPH3A antibodies be used to investigate neurodegenerative diseases?

RPH3A has been implicated in several neurodegenerative conditions:

• Huntington's disease: Deletion of RPH3A may be responsible for synaptic dysfunction in transgenic mouse models (R6/1)

• Alzheimer's disease: RPH3A is involved in pathological processes

• Pharmacological dyskinesias: RPH3A plays a role in the development of these movement disorders

Research approaches include:

• Comparing RPH3A expression levels in patient samples versus controls

• Investigating RPH3A localization changes in disease models

• Studying interactions between RPH3A and disease-associated proteins

• Examining phosphorylation states of RPH3A in pathological conditions

• Assessing the effects of RPH3A modulation on disease progression

What are the methodological considerations for studying RPH3A in neuroendocrine disorders?

When investigating RPH3A in neuroendocrine disorders such as central diabetes insipidus:

• Sample collection timing: Samples for anti-rabphilin-3A antibody measurement should be collected at specific timepoints, such as at the beginning of hypertonic saline infusion tests

• Antibody detection method: Western blotting using recombinant human rabphilin-3A protein lysate as antigen and patient serum as primary antibody is recommended

• Control samples: Include appropriate controls:

  • Positive controls from confirmed LINH cases

  • Negative controls from healthy individuals

  • Disease controls from other pituitary disorders

• Quantification: Objective scoring systems should be developed to classify antibody positivity based on band intensity

• Clinical correlation: Antibody results should be correlated with clinical findings, imaging data, and other laboratory parameters

How can RPH3A antibodies be used to study synaptic plasticity mechanisms?

RPH3A's role in regulating NMDA receptor stability makes it an important target for synaptic plasticity research:

• Localization studies:

  • Use double immunofluorescence labeling with RPH3A antibodies and synaptic markers (PSD-95, synaptophysin)

  • Employ super-resolution microscopy to visualize precise subsynaptic localization

• Activity-dependent changes:

  • Examine RPH3A distribution and phosphorylation after inducing long-term potentiation (LTP) or depression (LTD)

  • Compare RPH3A levels in high- versus low-activity neurons

• Protein complex analysis:

  • Use co-immunoprecipitation with RPH3A antibodies to isolate native protein complexes

  • Identify activity-dependent changes in binding partners

• Functional studies:

  • Combine electrophysiological recordings with molecular manipulation of RPH3A

  • Correlate RPH3A levels with changes in NMDAR-dependent currents and plasticity

Key methodological considerations include the use of phospho-specific antibodies to track activity-dependent modifications and careful preservation of native protein interactions during sample preparation.

What emerging applications of RPH3A antibodies show promise for neurological research?

Several innovative applications are emerging:

• Single-molecule localization microscopy: Using super-resolution techniques with RPH3A antibodies to map precise subsynaptic distribution

• Multiplexed imaging: Combining RPH3A antibodies with other synaptic markers in multiplexed immunofluorescence to create comprehensive synaptic "fingerprints"

• Live imaging: Developing strategies to visualize RPH3A dynamics in living neurons using antibody fragments or nanobodies

• Targeted therapeutics: Using RPH3A antibodies to deliver drugs or genetic material to specific neuronal populations

• Biomarker development: Further validating anti-RPH3A antibodies as diagnostic markers for neuroendocrine disorders beyond LINH

These approaches may provide new insights into neurological conditions and potential therapeutic targets.

How might novel RPH3A antibody engineering improve research capabilities?

Advances in antibody engineering offer new possibilities:

• Single-domain antibodies: Developing smaller antibody fragments that maintain specificity but with improved tissue penetration

• Bispecific antibodies: Creating antibodies that simultaneously target RPH3A and another protein of interest to study protein-protein interactions

• Conditionally active antibodies: Designing antibodies that become active only under specific cellular conditions

• Intrabodies: Engineering antibodies that function inside living cells to track or modulate RPH3A in real-time

• Site-specific conjugation: Developing precisely labeled antibodies for quantitative imaging or proximity-based assays