ophA1 Antibody

What is OPHN1 and why is it important for neuroscience research?

OPHN1 (Oligophrenin-1) is a Rho-GTPase-activating protein (Rho-GAP) that stimulates GTP hydrolysis of members of the Rho family. Its action on RHOA activity and signaling is implicated in the growth and stabilization of dendritic spines, making it critical for synaptic function . OPHN1 plays a key role in the activity-dependent maturation and plasticity of excitatory synapses by controlling their structural and functional stability . Mutations in the OPHN1 gene have been associated with X-linked mental retardation, cerebellar hypoplasia, and distinctive facial features, highlighting its importance in normal brain development and function . The protein is highly expressed in the brain, particularly in neurons of major regions including the hippocampus and cortex, where it is present in both axons and dendrites of principal neurons .

Which applications are validated for commercial OPHN1 antibodies?

Commercial OPHN1 antibodies have been validated for several experimental applications. Rabbit polyclonal OPHN1 antibodies are typically suitable for Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P) . Some antibodies have also been validated for immunocytochemistry-immunofluorescence (ICC-IF) . When selecting an OPHN1 antibody, researchers should verify the specific applications for which each product has been validated. For instance, the ab229655 antibody has been tested and confirmed effective for WB at 1/1000 dilution and IHC-P at 1/100 dilution with human samples . Other antibodies, such as HPA002919, are recommended for IHC at dilutions between 1:200-1:500 .

What is the typical protein localization pattern observed with OPHN1 antibodies?

OPHN1 demonstrates a distinctive subcellular localization pattern that is activity-dependent. Under normal conditions, OPHN1 is more than twofold enriched in dendritic spines compared to dendritic shafts in hippocampal neurons . This spine enrichment is dependent on synaptic activity and NMDA receptor activation, as pharmacological inhibition of neuronal activity with tetrodotoxin (TTX) or blockade of NMDA receptors with APV significantly reduces OPHN1's spine localization . In immunohistochemical analyses, OPHN1 antibodies typically detect the protein in tissues such as colon cancer and gastric cancer samples, with localization patterns that correspond to its function in particular cell types .

How should researchers design experiments to study OPHN1's role in synaptic plasticity?

When investigating OPHN1's role in synaptic plasticity, researchers should consider both electrophysiological and morphological approaches. Electrophysiological studies can employ simultaneous recordings of evoked excitatory post-synaptic currents (eEPSCs) from neurons with manipulated OPHN1 expression alongside control neurons . This allows direct comparison of AMPA receptor-mediated and NMDA receptor-mediated transmission. For temporal manipulation of OPHN1 expression, lentiviral vectors expressing OPHN1 shRNAs (targeting either translated regions or 3'-UTR) can be used to reduce endogenous OPHN1 levels .

For morphological studies, researchers should examine both spine density and size changes in response to OPHN1 manipulation. Two-photon laser scanning microscopy (TPLSM) of neurons expressing fluorescent proteins alongside OPHN1 constructs can reveal changes in spine morphology over time . A comprehensive experimental design would include both gain-of-function (OPHN1 overexpression) and loss-of-function (OPHN1 knockdown) approaches, with appropriate rescue experiments using RNAi-resistant OPHN1 constructs to confirm specificity .

What controls should be included when using OPHN1 antibodies for immunohistochemistry?

When performing immunohistochemistry with OPHN1 antibodies, researchers should include several critical controls:

• Positive tissue controls: Include tissues known to express OPHN1, such as brain tissue (particularly hippocampus and cortex) and certain cancer tissues where OPHN1 expression has been confirmed .

• Negative controls: Include tissues where OPHN1 expression is minimal or absent, or perform staining without the primary OPHN1 antibody to assess non-specific binding of the secondary antibody.

• Peptide competition assay: Pre-incubate the OPHN1 antibody with the immunizing peptide before application to tissue sections to confirm specificity.

• OPHN1 knockdown samples: When possible, include tissues or cells where OPHN1 expression has been reduced through genetic approaches (e.g., shRNA) to validate antibody specificity .

• Dilution series: Test a range of antibody dilutions (e.g., 1:100, 1:200, 1:500) to determine optimal staining conditions that maximize specific signal while minimizing background .

What are the recommended protocols for detecting OPHN1 expression in cancer tissues?

For detecting OPHN1 expression in cancer tissues, researchers should follow these methodological steps:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thickness) mounted on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), typically for 15-20 minutes at 95-100°C.

• Antibody incubation: Apply primary OPHN1 antibody at the recommended dilution (typically 1:100 for IHC-P) and incubate overnight at 4°C or for 1 hour at room temperature .

• Detection system: Use an appropriate detection system (e.g., HRP-conjugated secondary antibody and DAB chromogen) following the manufacturer's protocol.

• Counterstaining: Apply hematoxylin counterstain to visualize cell nuclei.

• Analysis: Evaluate OPHN1 expression patterns in relation to clinicopathological parameters. In prostate cancer studies, for example, OPHN1 overexpression has been related to high Gleason scores and poor prognosis .

How can researchers investigate the relationship between OPHN1 and androgen receptor signaling in cancer?

To investigate the relationship between OPHN1 and androgen receptor (AR) signaling in cancer, researchers should employ a multi-faceted approach:

• Gene amplification analysis: Analyze OPHN1 and AR copy number variations in cancer samples using quantitative PCR or next-generation sequencing, as OPHN1 is located in chromosome X near the AR gene and may be co-amplified .

• Expression correlation studies: Examine the correlation between OPHN1 and AR expression in patient samples using immunohistochemistry with validated antibodies for both proteins.

• Androgen deprivation experiments: Culture androgen-sensitive cancer cell lines (e.g., LNCaP, 22RV1) under androgen deprivation therapy (ADT) conditions and monitor OPHN1 expression changes using Northern blotting or qRT-PCR .

• Functional studies: Manipulate OPHN1 expression in cancer cells through transfection with OPHN1 overexpression vectors or siRNA/shRNA knockdown constructs, then assess effects on cell viability, apoptosis, and migration under normal and ADT conditions .

• In vivo models: Create xenograft models by injecting cancer cells with modified OPHN1 expression into immunodeficient mice, then monitor tumor growth rates with and without ADT .

This comprehensive approach can reveal how OPHN1 amplification contributes to ADT resistance and cancer progression, potentially identifying new therapeutic targets for hormone-refractory cancers.

What are the challenges in distinguishing the Rho-GAP-dependent and independent functions of OPHN1?

Distinguishing between Rho-GAP-dependent and independent functions of OPHN1 presents several methodological challenges:

• Mutant design: Researchers must generate precise OPHN1 mutants that selectively abolish Rho-GAP activity without affecting other protein domains. The OPHN1-GAP mutant, which virtually lacks Rho-GAP activity, is crucial for these studies .

• Rescue experiments: Both wild-type OPHN1 (OPHN1-WT) and OPHN1-GAP mutant should be tested in rescue experiments to determine which functions depend on Rho-GAP activity. For example, OPHN1-WT, but not OPHN1-GAP, can rescue defects in AMPAR-mediated transmission caused by OPHN1 knockdown .

• Signaling pathway analysis: Researchers must assess the activation status of Rho GTPases and downstream effectors (e.g., ROCK, mDia) in the presence of OPHN1-WT versus OPHN1-GAP to identify Rho-dependent processes.

• Structure-function analysis: Beyond the Rho-GAP domain, OPHN1 contains other functional domains including a BAR domain, a PH domain, and proline-rich regions. The contribution of each domain should be systematically assessed through deletion or point mutations .

• Subcellular localization studies: While OPHN1's spine enrichment is not dependent on its Rho-GAP activity, the PH domain appears critical for proper localization. An OPHN1 mutant lacking the N-terminal PH domain localizes primarily to the nucleus rather than dendritic spines .

Understanding these distinct molecular mechanisms is essential for developing targeted therapeutic approaches for OPHN1-associated disorders.

How can OPHN1 antibodies be used to investigate the molecular mechanisms of X-linked mental retardation?

OPHN1 antibodies provide powerful tools for investigating the molecular mechanisms underlying X-linked mental retardation (XLMR):

• Patient sample analysis: OPHN1 antibodies can be used to assess protein expression and localization in post-mortem brain samples from patients with XLMR, particularly those with cerebellar hypoplasia, which is a hallmark of OPHN1 mutations .

• Animal model validation: In mouse models of OPHN1 deficiency that recapitulate behavioral, social, and cognitive impairments seen in humans, OPHN1 antibodies can confirm protein knockout/knockdown and examine compensatory changes in related signaling pathways .

• Synaptic function studies: OPHN1 antibodies can be used in immunofluorescence studies to track changes in protein localization during synaptic activity. This is particularly relevant as OPHN1 recruitment to dendritic spines is activity-dependent and requires NMDA receptor activation .

• Biochemical interaction studies: Co-immunoprecipitation with OPHN1 antibodies can identify protein interaction partners in the brain, revealing how OPHN1 mutations disrupt critical protein complexes at synapses.

• Developmental expression analysis: Immunohistochemistry with OPHN1 antibodies can map the spatiotemporal expression pattern during brain development, providing insights into when and where OPHN1 deficiency might impair normal neurodevelopment.

These approaches collectively help elucidate how OPHN1 mutations lead to cognitive impairment and cerebellar hypoplasia, potentially identifying therapeutic targets for intervention.

How should researchers address non-specific binding when using OPHN1 antibodies in Western blotting?

When encountering non-specific binding with OPHN1 antibodies in Western blotting, researchers should implement the following troubleshooting strategies:

• Optimize blocking conditions: Test different blocking agents (5% non-fat dry milk, 5% BSA, or commercial blocking buffers) and extend blocking time to 2 hours at room temperature or overnight at 4°C.

• Adjust antibody concentration: Titrate the primary OPHN1 antibody concentration. For example, ab229655 shows optimal results at 1/1000 dilution for Western blotting of A549 cell lysates . Higher dilutions may reduce non-specific binding while maintaining specific signal.

• Increase washing stringency: Extend wash steps with TBST (TBS with 0.1-0.3% Tween-20) to 4-5 washes of 10 minutes each after both primary and secondary antibody incubations.

• Validate with controls: Include lysates from cells with OPHN1 knockdown or overexpression as negative and positive controls, respectively. The predicted band size for OPHN1 is approximately 92 kDa .

• Use more specific detection methods: Consider using more sensitive and specific detection systems such as enhanced chemiluminescence (ECL) with lower background.

• Pre-adsorb antibody: If cross-reactivity is a concern, pre-adsorb the OPHN1 antibody with cell/tissue lysates from species or tissues known to generate non-specific bands.

What strategies can improve detection sensitivity when studying low-abundance OPHN1 in neuronal subpopulations?

To improve detection sensitivity for low-abundance OPHN1 in specific neuronal subpopulations, researchers should consider these methodological approaches:

• Signal amplification techniques: Employ tyramide signal amplification (TSA) or other enzymatic amplification methods to enhance detection sensitivity in immunohistochemistry and immunofluorescence.

• Antigen retrieval optimization: Test multiple antigen retrieval methods (heat-induced with various buffers, enzymatic with proteinase K or trypsin) to maximize epitope exposure while preserving tissue morphology.

• Combined fluorescent labeling: Use multi-color immunofluorescence to simultaneously label OPHN1 with markers of specific neuronal subpopulations, facilitating identification of cells with low OPHN1 expression.

• Super-resolution microscopy: Implement advanced imaging techniques such as STED (Stimulated Emission Depletion) or STORM (Stochastic Optical Reconstruction Microscopy) to detect and precisely localize low-abundance OPHN1 in subcellular compartments.

• Proximity ligation assay (PLA): Apply PLA to detect OPHN1 interactions with binding partners, which can amplify signal and provide information about protein-protein interactions in situ.

• Laser capture microdissection: Isolate specific neuronal populations for enriched protein extraction followed by more sensitive biochemical detection methods.

These approaches can significantly enhance the detection of low-abundance OPHN1, enabling more precise characterization of its expression patterns and functional interactions in diverse neuronal subpopulations.

How should researchers interpret conflicting OPHN1 expression data between different experimental techniques?

When faced with conflicting OPHN1 expression data from different experimental techniques, researchers should implement a systematic analytical approach:

• Technique-specific considerations: Recognize that each technique has inherent limitations. For example, immunohistochemistry provides spatial information but may be less quantitative than Western blotting, which can't provide cellular localization data.

• Antibody validation status: Evaluate whether the antibodies used have been properly validated for each specific application. Some antibodies perform well in Western blotting but poorly in immunohistochemistry or vice versa .

• Epitope accessibility: Consider that protein conformation, fixation methods, or protein-protein interactions may mask epitopes in certain techniques, leading to false-negative results.

• Isoform-specific detection: Determine whether the conflicting results might be due to differential detection of OPHN1 isoforms. Verify which protein regions are recognized by each antibody.

• Quantification methodology: Review the quantification methods used. For instance, relative protein levels in Western blotting depend heavily on loading controls and normalization strategies.

• Biological context: Consider that OPHN1 expression may genuinely differ between experimental contexts due to activity-dependent regulation, developmental stages, or disease states .

To resolve conflicts, researchers should perform additional validation experiments, such as using multiple antibodies recognizing different OPHN1 epitopes or complementary techniques like mRNA analysis (qRT-PCR, RNA-seq) to corroborate protein expression data.

What statistical approaches are recommended when analyzing OPHN1 expression in relation to clinical outcomes?

When analyzing OPHN1 expression in relation to clinical outcomes, researchers should employ these statistical approaches:

How might emerging antibody technologies enhance OPHN1 research beyond traditional applications?

Emerging antibody technologies offer promising avenues to advance OPHN1 research beyond conventional applications:

• Single-domain antibodies (nanobodies): These smaller antibody fragments can access epitopes that traditional antibodies cannot reach, potentially revealing new aspects of OPHN1 structure and function in living cells.

• Intrabodies: Genetically encoded antibody fragments that can be expressed inside cells to track and potentially manipulate OPHN1 in real-time, offering insights into dynamic regulation of OPHN1 localization during synaptic activity .

• BiTE (Bispecific T-cell Engager) technology: For cancer research, bispecific antibodies targeting both OPHN1 and T-cell markers could help explore OPHN1's role in tumor immunology, particularly in prostate cancers where OPHN1 amplification has been observed .

• Antibody-drug conjugates (ADCs): These could be developed to specifically target cancer cells with OPHN1 amplification, potentially offering therapeutic strategies for androgen-independent prostate cancers .

• Degrader antibodies: Antibodies engineered to induce selective degradation of OPHN1 could serve as research tools to achieve rapid, reversible protein knockdown without genetic manipulation.

• Spatially-resolved antibody-based proteomics: Technologies like Digital Spatial Profiling or CODEX can map OPHN1 expression in tissue contexts with unprecedented spatial resolution, revealing expression patterns in complex tissues.

These emerging technologies promise to overcome current limitations in studying OPHN1 biology and may lead to novel therapeutic approaches for both neurological disorders and cancers associated with OPHN1 dysfunction.

What research questions remain unanswered regarding OPHN1's dual role in neurological disorders and cancer?

Several critical research questions remain unanswered regarding OPHN1's dual involvement in neurological disorders and cancer:

• Mechanistic divergence: How do the molecular mechanisms of OPHN1 function differ between neurons and cancer cells? Does the Rho-GAP activity have distinct downstream targets in these different cellular contexts?

• Isoform-specific functions: Are there tissue-specific OPHN1 isoforms that might explain its diverse roles across different diseases? How do these isoforms differ in their interaction partners and subcellular localization?

• Therapeutic targeting: Can OPHN1 be targeted therapeutically in cancers without adversely affecting neuronal function? Is there a therapeutic window that might allow selective inhibition in cancer cells?

• Biomarker potential: Can OPHN1 expression or post-translational modifications serve as biomarkers for treatment response in cancer or disease progression in neurological disorders?

• Developmental regulation: How is OPHN1 expression and function regulated throughout development, and how might this inform our understanding of both neurodevelopmental disorders and cancer pathogenesis?

• Sex-specific effects: Given OPHN1's location on the X chromosome, are there sex-specific differences in its function or in the manifestation of OPHN1-related disorders that might inform personalized medicine approaches?

Addressing these questions will require interdisciplinary approaches combining neuroscience, cancer biology, and precision medicine, potentially leading to novel therapeutic strategies for both conditions.