OPHN1 Antibody

What is the OPHN1 protein and what are its primary biological functions?

OPHN1 (Oligophrenin-1) is a Rho-GTPase activating protein encoded by the OPHN1 gene located in the Xq12 region of the X chromosome. It stimulates GTP hydrolysis of members of the Rho family and plays critical roles in:

• Growth and stabilization of dendritic spines

• Synaptic function through regulation of RHOA activity and signaling

• Stabilization of AMPA receptors at postsynaptic sites

• Regulation of synaptic vesicle endocytosis at presynaptic terminals

• Localization of NR1D1 to dendrites, suppressing its repressor activity and protecting it from proteasomal degradation

The protein is expressed in both glial cells and neurons, where it colocalizes with F-actin, particularly at the tips of rising dendrites and at synapses. OPHN1 is ubiquitously expressed in fetal and adult brain tissue, with particularly high expression in the hippocampus, cerebellum, and olfactory bulb .

What applications are OPHN1 antibodies validated for in research?

OPHN1 antibodies have been validated for several common laboratory applications:

Typical working dilutions include 1/1000 for Western blot and 1/100 for immunohistochemical analysis, though optimal concentrations should be determined empirically for each experimental protocol .

What sample types are compatible with OPHN1 antibodies?

Based on available research data, OPHN1 antibodies have demonstrated reactivity with:

• Human samples (most extensively validated)

• Potential cross-reactivity with other mammalian species may be expected due to sequence homology, though specific validation is recommended before use with non-human samples

When using OPHN1 antibodies with tissue samples, standard fixation protocols with paraformaldehyde or formalin have proven effective for immunohistochemical applications .

How should samples be prepared for optimal OPHN1 antibody detection?

For optimal results when working with OPHN1 antibodies:

For Western blotting:

• Extract proteins using RIPA buffer containing protease inhibitors

• Determine protein concentration using BCA or similar assay

• Load 20 µg of protein per lane on 8-10% SDS-polyacrylamide gels

• Transfer to nitrocellulose membranes

• Block with 5% BSA for 1 hour at room temperature

• Use recommended dilutions (typically 1:3000) of OPHN1 antibody with overnight incubation at 4°C

• Detect using appropriate HRP-conjugated secondary antibody and ECL detection system

For immunohistochemistry:

• Use paraffin-embedded or frozen tissue sections

• Perform standard antigen retrieval (method should be optimized)

• Block with appropriate serum

• Incubate with OPHN1 antibody at recommended dilution (typically 1:100)

• Visualize using appropriate detection system

What controls should be included when validating OPHN1 antibody specificity?

When validating OPHN1 antibody specificity, the following controls are essential:

Positive controls:

• Cell lines with known OPHN1 expression (e.g., A549 human lung carcinoma cells)

• Tissue samples with established OPHN1 expression (e.g., brain tissue, particularly cerebellum and hippocampus)

Negative controls:

• Primary antibody omission control

• Isotype-matched irrelevant antibody control

• Cells/tissues with OPHN1 knockdown or knockout (if available)

• Blocking peptide competition assay using the immunogenic peptide

Additional validation:

• Verification of the expected molecular weight band (92 kDa) in Western blots

• Confirmation of appropriate subcellular localization pattern

• Cross-validation with alternative OPHN1 antibody clones

How can OPHN1 expression be quantified in Western blot analysis?

For accurate quantification of OPHN1 expression by Western blot:

• Include appropriate housekeeping protein controls (e.g., GAPDH) for normalization

• Use standardized protein loading (20 µg per lane recommended in published protocols)

• Ensure linearity of signal detection through preliminary experiments with varying protein concentrations

• Capture images using a digital chemiluminescence detection system

• Analyze band intensity using image analysis software (e.g., ImageJ)

• Calculate relative expression as the ratio of OPHN1 to loading control intensity

• Perform at least three independent experiments for statistical validity

• Include known positive controls and calibration standards where possible

Signal quantification can be performed using software like ImageJ (version 1.8.0; National Institutes of Health) as demonstrated in published research .

What are the best fixation and antigen retrieval methods for OPHN1 immunohistochemistry?

Based on published protocols, successful OPHN1 immunohistochemistry has been achieved using:

Fixation:

• Standard formalin fixation (10% neutral buffered formalin)

• Paraformaldehyde fixation (4%) for fresh tissue samples

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• For paraffin-embedded samples, deparaffinization followed by rehydration and HIER

While specific optimization may be required for different tissue types, these methods have been successfully employed in published research examining OPHN1 expression in tissues including colon cancer and gastric cancer samples .

How should experiments be designed to study OPHN1 in neurological disorders?

When designing experiments to study OPHN1 in neurological disorders, consider the following approach:

• Subject selection:

  • Include patients with defined phenotypes associated with OPHN1 mutations (e.g., XLID, cerebellar hypoplasia)

  • Select appropriate age-matched and sex-matched controls

  • Consider family-based studies where possible

• Genetic characterization:

  • Use MLPA analysis to detect deletions in the OPHN1 gene

  • Consider whole-exome sequencing to identify point mutations

  • Verify mutations with Sanger sequencing

• Protein expression analysis:

  • Compare OPHN1 protein levels in patient-derived samples

  • Use validated OPHN1 antibodies for Western blotting or immunohistochemistry

  • Quantify differences in expression and localization

• Functional studies:

  • Develop cellular models using patient-derived cells or CRISPR-engineered cell lines

  • Assess dendritic spine morphology and synaptic function

  • Evaluate Rho-GTPase activity in mutant vs. wild-type conditions

• Neuroimaging correlation:

  • Correlate molecular findings with brain imaging results (MRI)

  • Focus on cerebellar hypoplasia and ventricular enlargement

This integrated approach has proven valuable in published studies investigating the role of OPHN1 in syndromic X-linked intellectual disability .

What experimental models are appropriate for studying OPHN1 functions?

Several experimental models have been successfully used to study OPHN1 functions:

Cellular Models:

• Neuronal cell lines (e.g., SH-SY5Y, primary neurons)

• Cancer cell lines for specific applications (e.g., A549, LNCaP, 22RV1, PC3)

• Patient-derived fibroblasts or iPSC-derived neurons

Animal Models:

• OPHN1 knockout or knockdown mouse models

• Xenograft models for cancer-related studies

• BALB/c NU/NU nude mice for in vivo experiments

Expression Systems:

• Recombinant lentiviral vectors for gene expression manipulation

• siRNA-mediated knockdown approaches

• CRISPR-Cas9 gene editing systems

The choice of model should align with the specific research question and consider the relevant pathophysiological context (e.g., neurodevelopmental disorders vs. cancer research) .

How can OPHN1 antibodies be used to investigate synaptic function in neurological disorders?

OPHN1 antibodies can be valuable tools for investigating synaptic function in neurological disorders through multiple approaches:

• Immunofluorescence co-localization studies:

  • Co-stain with OPHN1 antibodies and markers for synaptic structures (e.g., PSD-95, synaptophysin)

  • Perform high-resolution confocal or super-resolution microscopy

  • Quantify co-localization using appropriate software tools

  • Compare patterns between control and disease samples

• Immunoprecipitation-based interaction studies:

  • Use OPHN1 antibodies to pull down protein complexes

  • Identify binding partners through mass spectrometry

  • Verify specific interactions using co-immunoprecipitation and Western blotting

  • Map interaction domains using truncated constructs

• Live-cell imaging:

  • Generate fluorescently tagged OPHN1 constructs

  • Monitor dynamics at synapses in response to activity

  • Compare wild-type and mutant forms of OPHN1

  • Correlate with electrophysiological recordings

• Electron microscopy:

  • Use immunogold labeling with OPHN1 antibodies

  • Determine precise ultrastructural localization at synapses

  • Compare distribution in control versus disease samples

These approaches can help elucidate how OPHN1 dysfunction contributes to synaptic abnormalities in conditions such as X-linked intellectual disability .

What are the current challenges in OPHN1 antibody research and how can they be addressed?

Several challenges exist in OPHN1 antibody research that researchers should be aware of:

Challenge 1: Antibody specificity

• Solution: Perform rigorous validation including Western blot confirmation of the expected 92 kDa band, knockdown controls, and cross-validation with multiple antibody clones

• Implement peptide competition assays to confirm epitope specificity

Challenge 2: Isoform detection

• Solution: Select antibodies with characterized epitopes relative to known OPHN1 isoforms

• Verify which isoforms are detected by PCR or other molecular methods before antibody-based studies

Challenge 3: Cross-reactivity in non-human species

• Solution: Perform preliminary validation studies in the species of interest

• Consider sequence homology analysis before application in new species

Challenge 4: Reproducibility across different tissue preparations

• Solution: Optimize fixation and antigen retrieval protocols for each tissue type

• Include standardized positive controls in each experiment

• Maintain consistent protocols across experiments for comparative studies

Challenge 5: Quantitative analysis standardization

• Solution: Develop robust quantification methods with appropriate normalization

• Use digital image analysis with standardized parameters

• Include calibration standards when possible

How are OPHN1 antibodies being used in cancer research, particularly in prostate cancer studies?

OPHN1 antibodies have become increasingly important in cancer research, with particular applications in prostate cancer studies:

• Expression analysis in ADT-treated samples:

  • OPHN1 antibodies are used to measure protein expression in androgen deprivation therapy (ADT) settings

  • Western blot analysis with OPHN1 antibodies (1:3,000 dilution) has revealed ADT-induced overexpression of OPHN1 in prostate cancer cell lines

  • Quantification of expression changes correlates with cancer progression markers

• Tissue microarray studies:

  • Immunohistochemical staining with OPHN1 antibodies in patient-derived samples

  • Correlation of expression levels with Gleason scores and clinical outcomes

  • Identification of OPHN1 as a potential prognostic marker

• Mechanistic studies:

  • Investigation of OPHN1's role in cancer cell survival and apoptosis resistance

  • Analysis of OPHN1 impact on cell migration using antibody-based detection methods

  • Correlation with AR signaling pathway components

• Therapeutic targeting evaluation:

  • Monitoring OPHN1 expression changes in response to novel therapeutics

  • Assessment of OPHN1 as a potential therapeutic target

  • Study of relationship between OPHN1 and drug resistance mechanisms

These applications have revealed that OPHN1 amplification contributes to prostate cancer progression and resistance to androgen deprivation therapy, suggesting potential new avenues for therapeutic intervention .

What are common issues in Western blotting with OPHN1 antibodies and how can they be resolved?

When performing Western blotting with OPHN1 antibodies, researchers may encounter several common issues:

For optimal results with OPHN1 antibodies in Western blotting, researchers should consider the specific protocol that has been validated, which includes RIPA buffer extraction, 8-10% SDS-PAGE gels, and overnight incubation at 4°C with 1:3000 antibody dilution .

How should contradictory results between OPHN1 antibody studies be interpreted and reconciled?

When faced with contradictory results between OPHN1 antibody studies, researchers should systematically evaluate potential sources of discrepancy:

• Antibody-specific factors:

  • Compare epitope locations between different antibodies

  • Assess validation methods used in each study

  • Consider potential isoform specificity differences

  • Evaluate secondary antibody compatibility and detection methods

• Experimental design variations:

  • Analyze differences in sample preparation protocols

  • Review buffer compositions and blocking reagents

  • Compare quantification methods and normalization approaches

  • Assess statistical analysis techniques

• Biological context differences:

  • Consider cell/tissue type variations

  • Evaluate disease state and treatment conditions

  • Assess species differences and developmental stages

  • Review potential confounding genetic or epigenetic factors

• Reconciliation approach:

  • Perform side-by-side comparison experiments

  • Implement orthogonal validation techniques (e.g., mRNA quantification)

  • Use genetic manipulation to create defined controls

  • Collaborate with authors of contradictory studies for direct protocol comparison

This structured analysis can help identify whether discrepancies arise from technical issues or reflect genuine biological complexity in OPHN1 expression and function across different experimental contexts .

What factors affect the reproducibility of OPHN1 immunohistochemistry results?

Several key factors can impact the reproducibility of OPHN1 immunohistochemistry results:

Tissue processing factors:

• Fixation method and duration (formalin overfixation can mask epitopes)

• Tissue section thickness (optimal: 4-5 μm)

• Antigen retrieval method and duration

• Storage conditions of paraffin blocks or slides

Technical factors:

• Antibody lot variation (validation of each new lot recommended)

• Antibody concentration (1:100 dilution typically effective)

• Incubation conditions (temperature and duration)

• Detection system sensitivity and batch variation

• Counterstaining intensity and method

Analysis factors:

• Subjective interpretation of staining intensity

• Region selection for quantification

• Image acquisition parameters

• Threshold selection for positive/negative discrimination

• Scorer training and blinding procedures

Biological factors:

• Patient heterogeneity

• Tissue heterogeneity within samples

• Disease stage and treatment effects

• Co-occurring genetic alterations

To maximize reproducibility, researchers should implement standardized protocols, include appropriate controls in each experiment, use digital image analysis where possible, and consider multi-observer scoring for critical studies .

How are advanced technologies enhancing OPHN1 antibody-based research?

Emerging technologies are significantly advancing OPHN1 antibody-based research:

Single-cell technologies:

• Mass cytometry (CyTOF) with OPHN1 antibodies enables high-dimensional protein analysis at single-cell resolution

• Single-cell Western blotting allows protein quantification in individual cells

• Imaging mass cytometry combines OPHN1 antibody detection with spatial information

Advanced microscopy:

• Super-resolution microscopy techniques (STORM, PALM, STED) provide nanoscale localization of OPHN1

• Expansion microscopy physically enlarges specimens for enhanced resolution

• Lightsheet microscopy enables rapid 3D imaging of large tissue volumes with minimal photobleaching

Multiplexed detection:

• Multiplexed immunofluorescence allows simultaneous detection of OPHN1 with multiple markers

• Cyclic immunofluorescence (CycIF) enables sequential antibody staining rounds

• Spatial transcriptomics correlates OPHN1 protein expression with mRNA profiles

Automation and high-throughput approaches:

• Automated immunohistochemistry platforms improve reproducibility

• Tissue microarrays enable simultaneous analysis of hundreds of samples

• Machine learning algorithms enhance quantitative analysis of staining patterns

These technological advances are enabling more comprehensive and quantitative studies of OPHN1 localization, interaction partners, and expression patterns in both neurological disorders and cancer research contexts .

What role might OPHN1 antibodies play in developing therapeutic approaches for OPHN1-related disorders?

OPHN1 antibodies are likely to contribute to therapeutic development for OPHN1-related disorders in several important ways:

• Target validation and mechanistic studies:

  • Antibody-based characterization of OPHN1 expression in patient samples helps validate therapeutic targets

  • Immunoprecipitation followed by mass spectrometry can identify interaction partners as potential drug targets

  • Tracking OPHN1 localization changes in response to experimental therapeutics

• Biomarker development:

  • OPHN1 antibodies enable quantification of protein expression as potential predictive or prognostic biomarkers

  • Assessment of OPHN1 phosphorylation or other post-translational modifications as response indicators

  • Companion diagnostic development for patient stratification

• Therapeutic antibody development:

  • Research antibodies provide foundation for developing function-modulating therapeutic antibodies

  • ADC (antibody-drug conjugate) strategies targeting OPHN1-expressing cells

  • Intrabody approaches to modulate OPHN1 function in specific cellular compartments

• Treatment response monitoring:

  • OPHN1 antibodies allow assessment of treatment effects on protein expression and localization

  • Correlation of OPHN1 levels with symptom improvement in clinical trials

  • Early detection of emerging resistance mechanisms

In prostate cancer research specifically, OPHN1 antibodies have already contributed to understanding ADT resistance mechanisms, potentially leading to combination therapy approaches targeting both AR signaling and OPHN1-mediated pathways .

How does OPHN1 research intersect with advances in antibody engineering and screening technologies?

The intersection of OPHN1 research with antibody engineering and screening technologies represents a frontier with significant potential:

• High-throughput antibody generation:

  • The oPool+ display platform combines oligo pool synthesis and mRNA display to rapidly screen native antibody pairs, potentially enabling generation of novel OPHN1-targeting antibodies with optimized properties

  • Active learning approaches for antibody-antigen binding prediction can accelerate OPHN1 antibody development by reducing the number of required experimental variants and speeding up the learning process

• Antibody engineering for enhanced properties:

  • Engineering OPHN1 antibodies with improved tissue penetration for brain delivery

  • Development of bispecific antibodies targeting OPHN1 plus complementary targets

  • Creating intrabodies designed to localize to specific subcellular compartments

  • Engineering antibody fragments with tailored binding properties

• Advanced screening methodologies:

  • Library-on-library approaches enable identification of specific OPHN1-interacting antibody candidates

  • Machine learning models that predict target binding by analyzing many-to-many relationships between antibodies and antigens can accelerate discovery

  • Out-of-distribution prediction capabilities improve selection of candidates likely to work in complex in vivo environments

• Translational applications:

  • Development of highly specific OPHN1 antibodies for improved diagnosis of OPHN1-related disorders

  • Engineering therapeutic antibodies or antibody-drug conjugates targeting OPHN1 in cancer applications

  • Creating imaging agents based on OPHN1 antibodies for visualization of expression patterns in vivo

These advances may both enhance basic research tools and accelerate development of clinical applications targeting OPHN1-related pathologies in neurological disorders and cancer .