Phospho-RPH3A (S237) Antibody

What is the functional significance of RPH3A phosphorylation at Serine 237?

RPH3A (Rabphilin-3A) phosphorylation at Serine 237 plays a critical role in regulating synaptic vesicle trafficking and calcium-triggered neurotransmitter release. The phosphorylation state at this specific residue modulates the protein's interaction with synaptic vesicle membranes and its ability to participate in the docking and fusion steps of regulated exocytosis . In the presynaptic compartment, RPH3A is recruited by RAB3A to synaptic vesicle membranes in a GTP-dependent manner, where phosphorylation at Ser237 can alter its binding affinity and regulatory functions . Additionally, in postsynaptic compartments, phosphorylated RPH3A contributes to NMDA receptor stability through formation of a ternary complex with GRIN2A and DLG4, highlighting its diverse functional roles based on phosphorylation status .

What are the optimal sample preparation methods for detecting phosphorylated RPH3A?

For optimal detection of phosphorylated RPH3A at Serine 237, samples should be processed with phosphatase inhibitors immediately after collection to preserve the phosphorylation state. The recommended protocol includes:

• Homogenize tissue samples in ice-cold RIPA buffer supplemented with protease inhibitors and phosphatase inhibitor cocktail.

• For cultured cells, lyse directly in the buffer after washing with cold PBS.

• Maintain samples at 4°C throughout processing to minimize phosphatase activity.

• Centrifuge lysates at 12,000×g for 15 minutes at 4°C and collect supernatants.

• Quantify protein concentration using a compatible assay (BCA or Bradford).

• For Western blot applications, denature samples in Laemmli buffer at 95°C for 5 minutes.

When detecting phospho-RPH3A, fresh samples generally yield better results than frozen ones, though flash-freezing in liquid nitrogen immediately after collection can preserve phosphorylation status reasonably well .

What dilution ranges are recommended for different experimental applications?

The following table summarizes optimal dilution ranges for Phospho-RPH3A (S237) antibody applications:

For all applications, the optimal working dilution should be determined empirically for each specific experimental condition and sample type .

How can I distinguish between different phosphorylation states of RPH3A in my experimental system?

Distinguishing between different phosphorylation states of RPH3A requires careful experimental design:

• Use phospho-specific antibodies targeting distinct sites (e.g., Ser237 vs. Ser234) in parallel experiments .

• Implement lambda phosphatase treatment as a negative control to confirm phospho-specificity.

• For comprehensive phosphorylation profiling, combine immunoblotting with mass spectrometry analysis.

• Consider using Phos-tag™ SDS-PAGE to separate proteins based on their phosphorylation status, which can resolve multiple phosphorylation states simultaneously.

• For functional validation, incorporate site-directed mutagenesis (S237A or S237D) to mimic non-phosphorylated or constitutively phosphorylated states, respectively.

When analyzing multiple phosphorylation sites, sequential immunoprecipitation with different phospho-specific antibodies can help delineate the relative abundance of distinct phosphorylated populations .

What are the critical controls needed when using Phospho-RPH3A (S237) antibody in neuronal studies?

When conducting neuronal studies with Phospho-RPH3A (S237) antibody, implement these essential controls:

• Phosphatase-treated negative control: Treat duplicate samples with lambda phosphatase to demonstrate phospho-specificity.

• Total RPH3A detection: Use a separate antibody against total RPH3A to normalize phospho-signal and account for expression level variations.

• RPH3A knockout/knockdown: Include samples from RPH3A knockout or knockdown models to confirm antibody specificity.

• Peptide competition assay: Pre-incubate antibody with the immunizing phosphopeptide to verify epitope-specific binding.

• Cross-reactivity assessment: Test against samples containing phosphorylated proteins with similar consensus sequences.

• Positive controls: Include samples known to contain high levels of phosphorylated RPH3A, such as stimulated neuronal cultures treated with PKC activators .

These controls collectively ensure that observed signals genuinely represent phosphorylated RPH3A at Serine 237 rather than non-specific binding or cross-reactivity.

How does sample preparation affect the detection of phosphorylated RPH3A in different subcellular compartments?

The detection of phosphorylated RPH3A across subcellular compartments is significantly influenced by sample preparation methods:

For membrane-associated phospho-RPH3A:

• Use gentle detergents (0.5% NP-40 or 0.1% Triton X-100) in extraction buffers

• Consider subcellular fractionation to isolate synaptic vesicles or postsynaptic density fractions

• Avoid harsh sonication that may disrupt membrane associations

For cytosolic phospho-RPH3A:

• Higher detergent concentrations (1% Triton X-100) improve extraction

• Ultracentrifugation (100,000×g) helps separate truly soluble fractions

For dendritic spine-localized phospho-RPH3A:

• Specialized synaptosomal preparation methods yield better results

• Consider using synaptic plasma membrane isolation protocols

The phosphorylation state of RPH3A affects its subcellular distribution, with phosphorylated Ser237 RPH3A showing enrichment in synaptic vesicle preparations versus total homogenates. This differential localization must be considered when designing experiments targeting specific subcellular pools .

What are common pitfalls in Western blot analysis of Phospho-RPH3A (S237) and how can they be addressed?

When performing Western blot analysis of Phospho-RPH3A (S237), researchers frequently encounter these challenges:

• Low signal intensity:

  • Reduce antibody dilution (try 1:500)

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Increase protein loading (50-80μg total protein)

• Non-specific bands:

  • Increase blocking time and concentration (5% BSA in TBST, 2 hours)

  • Optimize washing steps (5× washes, 5 minutes each)

  • Use 0.5% Tween-20 in washing buffer

  • Pre-adsorb antibody with non-relevant tissue lysate

• Inconsistent phosphorylation signal:

  • Add phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate)

  • Keep samples consistently on ice

  • Reduce time between tissue collection and protein extraction

  • Standardize stimulation protocols for consistent phosphorylation

• Degradation products:

  • Add protease inhibitor cocktail to all buffers

  • Process samples immediately or flash-freeze

  • Avoid repeated freeze-thaw cycles of prepared samples

How can I validate that my antibody is specifically detecting the phosphorylated form of RPH3A?

To validate the phospho-specificity of your RPH3A (S237) antibody, implement this comprehensive validation workflow:

• Parallel detection strategy:

  • Run identical samples on two blots

  • Probe one with phospho-specific antibody

  • Probe the second with total RPH3A antibody

  • Compare band patterns and intensity ratios

• Phosphatase treatment experiment:

  • Divide your sample into two aliquots

  • Treat one with lambda protein phosphatase (400 units/100μg protein, 30 minutes at 30°C)

  • The phospho-specific signal should disappear in treated samples while total RPH3A remains detectable

• Stimulation response:

  • Treat cells/tissues with PKC activators (phorbol esters, 100nM, 15-30 minutes)

  • The phospho-S237 signal should increase while total RPH3A remains constant

• Peptide competition:

  • Pre-incubate antibody with the immunizing phosphopeptide (10-100× molar excess)

  • The specific signal should be abolished

• Mass spectrometry correlation:

  • Confirm phosphorylation at S237 by mass spectrometry analysis of immunoprecipitated RPH3A

  • Compare with antibody-based detection methods for concordance

What experimental factors might affect the phosphorylation state of RPH3A at Serine 237?

The phosphorylation state of RPH3A at Serine 237 is dynamically regulated by multiple factors that should be carefully controlled in experimental designs:

• Neuronal activity modulation:

  • Depolarizing conditions (KCl 50mM, 10 minutes) can significantly increase phosphorylation

  • Synaptic activity blockade (TTX 1μM, 2-4 hours) reduces phosphorylation levels

• Calcium signaling:

  • Intracellular calcium chelation (BAPTA-AM 10μM, 30 minutes) decreases phosphorylation

  • Calcium ionophores (ionomycin 2μM, 15 minutes) enhance phosphorylation

• Kinase/phosphatase balance:

  • PKC activators (PMA 100nM, 30 minutes) increase S237 phosphorylation

  • Calcineurin inhibitors (cyclosporin A 5μM, 1 hour) preserve phosphorylation

  • PKC inhibitors (Gö6983 1μM, 1 hour) reduce phosphorylation

• Metabolic state:

  • Glucose deprivation rapidly decreases phosphorylation (within 30 minutes)

  • ATP depletion conditions reduce phosphorylation levels

• Sample handling:

  • Post-mortem delay increases dephosphorylation (significant after 10 minutes at room temperature)

  • Hypoxic conditions accelerate dephosphorylation

Controlling these variables is critical for reproducible results, especially when comparing phosphorylation states across experimental conditions .

How can Phospho-RPH3A (S237) antibody be used to investigate synaptic plasticity mechanisms?

Phospho-RPH3A (S237) antibody offers several advanced approaches for investigating synaptic plasticity mechanisms:

• Temporal phosphorylation profiling during LTP/LTD induction:

  • Collect samples at defined timepoints following plasticity induction

  • Quantify phospho-RPH3A:total RPH3A ratios to track signaling cascades

  • Correlate phosphorylation dynamics with electrophysiological measurements

• Spatial mapping of phosphorylation in neuronal compartments:

  • Use super-resolution microscopy with phospho-specific antibodies

  • Examine redistribution of phosphorylated RPH3A during plasticity events

  • Co-localize with synaptic markers to assess compartment-specific regulation

• Activity-dependent phosphorylation analysis:

  • Combine with optogenetic stimulation of specific neural circuits

  • Measure phosphorylation changes in response to defined activity patterns

  • Correlate with synaptic release probability measurements

• NMDA receptor complex interaction studies:

  • Use proximity ligation assay with phospho-RPH3A and NMDAR subunits

  • Investigate how phosphorylation alters the formation of the RPH3A-GRIN2A-DLG4 complex

  • Determine effects on NMDAR localization and stability

These approaches can reveal how RPH3A phosphorylation contributes to the molecular mechanisms underlying synaptic strength modulation and memory formation .

What are the most effective protocols for multiplexed detection of RPH3A phosphorylation states alongside other synaptic phosphoproteins?

For effective multiplexed detection of RPH3A phosphorylation states alongside other synaptic phosphoproteins, consider these optimized protocols:

• Sequential immunoblotting protocol:

  • Use primary antibodies from different host species (rabbit anti-phospho-RPH3A with mouse anti-phospho-synapsin)

  • Strip and reprobe membranes (optimized stripping buffer: 200mM glycine, 0.1% SDS, 1% Tween-20, pH 2.2)

  • Verify complete stripping using secondary antibody alone

  • Document order-dependent effects by alternating antibody application sequence

• Multiplexed immunofluorescence:

  • Use primary antibodies from different species

  • Employ spectrally distinct fluorophore-conjugated secondaries

  • Apply tyramide signal amplification for low-abundance phosphoproteins

  • Include single-stain controls to verify antibody specificity

• Multi-parameter flow cytometry for synaptosomes:

  • Isolate synaptosomes using Percoll gradient centrifugation

  • Fix and permeabilize with 0.1% saponin

  • Label with fluorophore-conjugated phospho-specific antibodies

  • Gate on synaptosome populations based on size/complexity

• Phosphoprotein enrichment strategy:

  • Use titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC)

  • Enrich phosphopeptides prior to immunodetection

  • Compare enriched fractions to total lysates

  • Quantify enrichment efficiency using phosphoprotein standards

How can phosphorylation of RPH3A at Serine 237 be manipulated for functional studies?

To manipulate RPH3A phosphorylation at Serine 237 for functional studies, researchers can implement these advanced approaches:

• Genetic manipulation strategies:

  • Generate phospho-mimetic mutants (S237D or S237E) that simulate constitutive phosphorylation

  • Create phospho-deficient mutants (S237A) that prevent phosphorylation

  • Use site-specific knock-in mutations to replace endogenous RPH3A

  • Express mutants in RPH3A-null background to avoid interference from endogenous protein

• Pharmacological manipulation:

  • Apply PKC activators (phorbol 12-myristate 13-acetate, 100nM) to increase phosphorylation

  • Use PKC inhibitors (bisindolylmaleimide, 1-5μM) to decrease phosphorylation

  • Target specific PKC isoforms using isoform-selective compounds

  • Manipulate upstream signaling cascades that converge on S237 phosphorylation

• Optogenetic and chemogenetic approaches:

  • Develop light-activated kinases that can be targeted to RPH3A

  • Design RPH3A constructs with chemically-induced dimerization domains for rapid recruitment to kinases/phosphatases

  • Use engineered phosphatases with temporal control to selectively dephosphorylate during defined time windows

• Functional readouts to assess effects:

  • Measure synaptic vesicle release using FM dyes or pHluorin-based reporters

  • Analyze synaptic transmission strength with electrophysiology

  • Track protein-protein interactions using FRET sensors

  • Visualize synaptic vesicle cycling dynamics using live imaging

These manipulation approaches, combined with appropriate readouts, can establish causal relationships between RPH3A phosphorylation and synaptic function .

What are the key considerations for optimizing immunohistochemistry protocols with Phospho-RPH3A (S237) antibody?

For optimal immunohistochemistry results with Phospho-RPH3A (S237) antibody, consider these critical parameters:

• Tissue fixation optimization:

  • Paraformaldehyde fixation (4%, 24 hours) preserves phospho-epitopes better than alcoholic fixatives

  • Perfusion fixation yields superior results compared to immersion fixation

  • Post-fixation time should be limited (4-8 hours) to prevent epitope masking

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0, 95°C, 20 minutes) works effectively

  • Compare with Tris-EDTA (pH 9.0) to determine optimal buffer conditions

  • Allow gradual cooling to room temperature (30 minutes) after heating

• Blocking and permeabilization:

  • Use 10% normal goat serum with 0.3% Triton X-100 in PBS (2 hours, room temperature)

  • For phospho-epitopes, add phosphatase inhibitors to blocking solution

  • BSA (3%) can be used as an alternative blocking agent

• Primary antibody incubation:

  • Recommended dilution range: 1:100-1:300 in blocking buffer

  • Extend incubation to 48 hours at 4°C for deeper tissue penetration

  • Use humidified chambers to prevent section drying

• Signal amplification considerations:

  • Biotin-streptavidin systems provide 3-5× signal enhancement

  • Tyramide signal amplification may be necessary for low abundance phospho-epitopes

  • Balance signal enhancement with background control

How can I quantitatively assess changes in RPH3A phosphorylation across different experimental conditions?

For rigorous quantitative assessment of RPH3A phosphorylation changes across experimental conditions, implement these methodological approaches:

• Western blot quantification strategy:

  • Use a dual-channel imaging system for simultaneous detection of phospho and total RPH3A

  • Calculate phospho:total ratio for each sample to normalize for expression differences

  • Include a standard curve of recombinant phosphorylated protein for absolute quantification

  • Report results as fold-change relative to baseline condition

• ELISA-based quantification:

  • Develop a sandwich ELISA with capture antibody against total RPH3A

  • Detect with phospho-specific antibody followed by enzyme-conjugated secondary

  • Generate standard curves using synthetic phosphopeptides

  • Optimal dilution for detection antibody: 1:10000

• Mass spectrometry approaches:

  • Use stable isotope labeling (SILAC or TMT) for accurate comparison between conditions

  • Target the specific phosphopeptide containing S237

  • Calculate stoichiometry of phosphorylation using the ratio of phosphorylated to non-phosphorylated peptides

  • Validate mass spectrometry findings with antibody-based methods

• Statistical considerations:

  • Perform power analysis to determine appropriate sample size (typically n≥5 per condition)

  • Use appropriate statistical tests (ANOVA with post-hoc tests for multiple conditions)

  • Report effect sizes along with p-values

  • Consider non-parametric tests if normality assumptions are violated

What are the recommended protocols for studying RPH3A phosphorylation in neurodevelopmental contexts?

When investigating RPH3A phosphorylation in neurodevelopmental contexts, these specialized protocols yield optimal results:

• Developmental timepoint analysis:

  • Collect samples at defined developmental stages (embryonic, early postnatal, mature)

  • Normalize phospho-RPH3A signal to total RPH3A for each timepoint

  • Use microdissection to isolate specific brain regions with different maturation timelines

  • Correlate phosphorylation changes with synaptogenesis markers

• Primary neuronal culture system:

  • Establish low-density cultures (25,000 cells/cm²) for single-cell resolution imaging

  • Collect samples at days in vitro (DIV) 3, 7, 14, and 21 to span developmental stages

  • Co-stain for developmental markers (MAP2, Tau, synapsin) to correlate with maturation

  • Use live cell imaging with transfected FRET-based phosphorylation reporters

• Tissue preparation considerations:

  • For embryonic tissue, use shorter fixation times (2-4 hours)

  • Adjust antibody concentration based on developmental stage (higher concentrations for early timepoints)

  • Use thinner sections for embryonic samples (10-15μm vs. 30-40μm for adult)

  • Include age-matched controls for all experimental manipulations

• Functional correlations:

  • Pair phosphorylation analysis with electrophysiological recordings

  • Measure synapse formation rate and correlate with phosphorylation dynamics

  • Assess activity-dependent phosphorylation changes at different developmental stages

  • Compare wild-type with genetic models of neurodevelopmental disorders