ORP3C Antibody

What is ORP3 and what are its primary cellular functions?

ORP3 (Oxysterol-binding protein-related protein 3), also known as OSBPL3 or OSBP3, is a phosphoinositide-binding protein that plays several critical roles in cellular function. It primarily associates with both cell and endoplasmic reticulum (ER) membranes, acting as a lipid transfer protein at membrane contact sites. ORP3 can bind to the ER membrane protein VAPA and recruit it to plasma membrane sites, effectively linking these intracellular compartments . This interaction creates a bridge between the ER and plasma membrane that facilitates lipid transport and signaling. ORP3 has significant roles in regulating the actin cytoskeleton, cell polarity, and cell adhesion processes through its involvement in RRAS signaling pathways . Additionally, it demonstrates binding preferences for specific phosphoinositides (particularly PI(3,4)P2 and PI(3,4,5)P3) and can also bind 25-hydroxycholesterol and cholesterol, suggesting its involvement in sterol trafficking and homeostasis .

What makes polyclonal ORP3 antibodies suitable for different experimental applications?

Polyclonal ORP3 antibodies offer significant advantages in experimental applications due to their recognition of multiple epitopes on the ORP3 protein. This characteristic provides enhanced sensitivity in detection across various experimental techniques. For example, the rabbit polyclonal ORP3 antibody (such as ab224212) is validated for immunohistochemistry on paraffin-embedded tissues (IHC-P), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) applications using human samples . The versatility stems from the antibody being raised against recombinant fragment protein within Human OSBPL3 amino acids 300-450 . This region contains multiple epitopes that remain accessible under different experimental conditions, allowing detection of the protein in its native conformation (ICC/IF), denatured state (WB), or after fixation and embedding (IHC-P). For research requiring identification of ORP3 in these diverse experimental contexts, polyclonal antibodies provide reliable detection without requiring multiple antibody types.

How should I optimize Western blot protocols for ORP3 detection in different cell types?

Optimizing Western blot protocols for ORP3 detection requires careful consideration of several parameters:

Sample Preparation:

• For adherent cells: Lyse cells directly in the culture dish using RIPA buffer supplemented with protease inhibitors

• For tissue samples: Homogenize in RIPA buffer (1:10 w/v) with protease inhibitors

• Include phosphatase inhibitors if investigating phosphorylation states

• Sonicate briefly (3-5 pulses) to shear DNA and reduce sample viscosity

Electrophoresis and Transfer Parameters:

• Load 20-40 μg of total protein per lane

• Use 8-10% SDS-PAGE gels (ORP3 is approximately 94-95 kDa)

• Transfer at 100V for 1 hour using standard transfer buffer with 20% methanol

Antibody Incubation:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary ORP3 antibody at 1:1000 dilution in 5% BSA/TBST overnight at 4°C

• Wash 3x with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody at 1:5000 in 5% milk/TBST for 1 hour

• Wash 4x with TBST, 5 minutes each before detection

Cell-Type Specific Considerations:
For endothelial or epithelial cells where ORP3 expression may be lower, increase protein loading to 50 μg and extend primary antibody incubation to 16-18 hours at 4°C with gentle agitation. For brain tissue samples, add an additional sonication step and extend transfer time to 90 minutes to ensure complete protein transfer.

What are the optimal fixation and permeabilization conditions for ORP3 immunofluorescence studies?

For successful immunofluorescence detection of ORP3, the preservation of both membrane structures and protein epitopes is crucial. The following protocol optimizes these conditions:

Fixation Options:

Permeabilization:

• For PFA-fixed cells: 0.1-0.2% Triton X-100 in PBS for 10 minutes

• For methanol-fixed cells: additional permeabilization is typically unnecessary

Blocking and Antibody Incubation:

• Block with 5% normal serum (from the species of secondary antibody) in PBS with 0.1% Triton X-100 for 1 hour

• Incubate with ORP3 primary antibody at 1:200-1:500 dilution in blocking solution overnight at 4°C

• Wash 3x with PBS, 5 minutes each

• Incubate with fluorophore-conjugated secondary antibody at 1:500 in blocking solution for 1 hour at room temperature in the dark

• Counterstain with DAPI (1:1000) during the final 10 minutes of secondary antibody incubation

Critical Considerations:
When studying ORP3's association with ER-plasma membrane contact sites, dual immunostaining with VAP-A markers is recommended using the methanol fixation protocol, which better preserves these delicate membrane structures while allowing visualization of the ORP3-VAPA interaction .

How can I effectively investigate ORP3's role in membrane contact sites using proximity ligation assays?

Proximity Ligation Assay (PLA) provides a powerful approach to visualize and quantify ORP3's interactions at membrane contact sites, particularly with its binding partner VAPA. This technique enables detection of protein interactions that occur within 40nm distance, making it ideal for studying membrane contact sites.

Experimental Protocol:

• Fix cells using 4% PFA for 15 minutes followed by 0.2% Triton X-100 permeabilization

• Block with Duolink blocking solution for 1 hour at 37°C

• Incubate with primary antibodies against ORP3 (rabbit polyclonal) and VAPA (mouse monoclonal) at 1:100 dilution overnight at 4°C

• Apply PLA probes (anti-rabbit PLUS and anti-mouse MINUS) for 1 hour at 37°C

• Perform ligation (30 minutes) and amplification (100 minutes) according to manufacturer's protocol

• Counterstain with DAPI and phalloidin for nuclear and actin visualization

Analysis Approach:

• Quantify PLA signals using confocal microscopy (minimum 20 cells per condition)

• Measure both signal intensity and spatial distribution relative to cell edges

• For statistical validity, analyze at least three independent experiments

Controls and Validation:

• Positive control: Known interaction partners (e.g., VAPA and VAPB)

• Negative control: Omission of one primary antibody

• Specificity control: siRNA knockdown of ORP3 to verify signal reduction

This method provides quantitative data on the ORP3-VAPA interaction at membrane contact sites, allowing assessment of how interventions (pharmacological treatments, gene knockdown, etc.) affect these interactions in live cells.

What strategies can address non-specific binding when using ORP3 antibodies in co-immunoprecipitation experiments?

Non-specific binding in co-immunoprecipitation (co-IP) experiments with ORP3 antibodies can significantly compromise data quality. The following strategies can minimize these issues:

Optimized Lysis and Binding Conditions:

• Use mild lysis buffers (e.g., 20mM HEPES pH 7.4, 150mM NaCl, 1% CHAPS or 0.5% NP-40) to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads (1 hour at 4°C) before adding ORP3 antibody

• Add 0.1-0.2% BSA to binding buffer to reduce non-specific interactions

Antibody Selection and Controls:

• Use monoclonal antibodies when available for higher specificity

• Include IgG isotype control from the same species as the ORP3 antibody

• Include a sample with ORP3 knockdown/knockout cells as negative control

Washing Optimization:
Implement a graduated washing strategy with increasing stringency:

• Two washes with lysis buffer

• Two washes with lysis buffer containing 300mM NaCl

• One final wash with PBS or TBS

Validation Approaches:

• Perform reverse co-IP (use antibody against suspected interacting partner)

• Validate interactions using proximity ligation assay

• Consider crosslinking approaches for transient interactions

When investigating ORP3-VAPA interactions specifically, include 1mM CaCl₂ in buffers to stabilize membrane-associated interactions and consider mild crosslinking (0.5-1% formaldehyde for 10 minutes at room temperature) before lysis to capture transient interactions at membrane contact sites.

How can computational approaches enhance antibody design for studying ORP3-related membrane interactions?

Recent advances in computational antibody design offer promising approaches for developing highly specific antibodies targeting ORP3 in membrane contact site research:

Computational Antibody Design Approaches:

• Inverse folding models like AbMPNN can generate new antibody sequences that maintain structural features compatible with specific ORP3 epitopes

• Protein language models such as ESM can guide mutations to improve binding affinity while enhancing developability properties

• Graph-based architectures like GearBind can predict the effect of mutations on antibody-antigen complexes, facilitating affinity maturation

Epitope-Focused Design Strategy:
For membrane-associated proteins like ORP3, targeting specific domains involved in protein-protein interactions requires carefully designed epitope selection:

• Identify accessible epitopes within the ORP3 lipid-binding domain (amino acids 300-450)

• Use computational models to design antibodies with minimal hydrophobic patches to reduce non-specific membrane binding

• Employ RFDiffusion or similar tools for structure-guided design, although success rates may vary depending on implementation

Validation and Screening Metrics:
After computational design, antibody candidates should be screened using:

• Size-exclusion chromatography (SEC) to assess aggregation propensity

• Differential scanning fluorimetry (DSF) for thermal stability

• Binding assays with both soluble and membrane-bound ORP3

This computational approach has demonstrated success rates of approximately 54% for generating binding antibodies in similar applications , making it a viable strategy for developing new tools to study ORP3 in complex membrane environments.

What advanced imaging techniques are most effective for studying ORP3 dynamics at membrane contact sites?

Understanding ORP3 dynamics at membrane contact sites requires specialized imaging approaches that overcome the limitations of conventional microscopy:

Super-Resolution Microscopy Approaches:

• STORM (Stochastic Optical Reconstruction Microscopy): Achieves 20-30nm resolution, ideal for visualizing ORP3 clustering at ER-plasma membrane contact sites

• PALM (Photoactivated Localization Microscopy): Effective when using photoactivatable fluorescent protein-tagged ORP3 constructs

• SIM (Structured Illumination Microscopy): Provides 100nm resolution with less technical complexity than STORM/PALM

Live-Cell Imaging Strategies:

• FRET-based approaches using ORP3-CFP and VAPA-YFP fusions to monitor protein interactions in real-time

• Split-GFP complementation assays where fragments are fused to ORP3 and VAPA, generating fluorescence only upon interaction

• Lattice light-sheet microscopy for extended imaging of ORP3 dynamics with minimal phototoxicity

Sample Preparation Considerations:

• For fixed samples: 4% PFA fixation followed by 0.1% glutaraldehyde post-fixation preserves membrane ultrastructure

• For live imaging: Expression levels of fluorescent protein fusions should be kept minimal (use weak promoters or inducible systems)

Quantitative Analysis Framework:

• Track ORP3-positive contact sites over time using particle tracking algorithms

• Measure dwell time, movement, and intensity changes of ORP3 at contact sites

• Correlate ORP3 dynamics with cellular events using multi-channel imaging

A combined approach using both super-resolution microscopy of fixed samples and live-cell imaging provides comprehensive insights into both the nanoscale organization and temporal dynamics of ORP3 at membrane contact sites. This multi-modal imaging strategy allows researchers to connect static structural information with dynamic functional data.

How might targeting ORP3 with specific antibodies reveal its role in disease models?

The strategic use of ORP3-specific antibodies in disease models represents a promising research direction with significant therapeutic implications:

Neurodegenerative Disease Applications:
Given ORP3's role in membrane contact sites and lipid transport, specific antibodies could be used to:

• Probe alterations in ER-plasma membrane contacts in Alzheimer's disease models

• Investigate dysregulation of cholesterol transport in neuronal cells

• Monitor changes in ORP3-VAPA complexes in relation to neuronal stress responses

Cancer Research Applications:

• Examine ORP3 involvement in altered cell adhesion and migration in metastatic models

• Investigate how aberrant RRAS signaling through ORP3-VAPA complexes affects integrin activation in cancer cells

• Develop antibody-based imaging tools to visualize ORP3 redistribution during epithelial-mesenchymal transition

Methodological Approaches:

• Development of conformation-specific antibodies that recognize active versus inactive ORP3 states

• Creation of antibodies that specifically block the ORP3-VAPA interaction

• Design of intrabodies that can track ORP3 in living cells without disrupting function

Therapeutic Potential:
While direct antibody therapeutics may be challenging due to the intracellular location of ORP3, research applications of these tools could reveal druggable nodes in ORP3-dependent pathways. Specifically, antibodies that can distinguish between cholesterol-bound and unbound states of ORP3 would provide valuable insights into how cellular cholesterol levels affect ORP3-dependent signaling in disease contexts.

What are the technical considerations for developing site-specific ORP3 antibodies that distinguish between its different functional states?

Developing antibodies that can distinguish between different functional states of ORP3 requires sophisticated technical approaches:

Epitope Selection Strategy:

• Target conformationally distinct regions that change upon ligand binding

• Focus on the phosphoinositide-binding pocket that undergoes conformational changes when binding PI(3,4)P2 versus PI(3,4,5)P3

• Consider epitopes at the ORP3-VAPA interface that are accessible only in the unbound state

Advanced Antibody Engineering Approaches:

• Employ adaptive autoregressive diffusion models that can be trained to recognize specific protein conformations

• Utilize pretrainable geometric graph neural networks that account for the three-dimensional structure of the antigenic site

• Apply contrastive learning techniques that differentiate between bound and unbound states

Validation Requirements:
A rigorous validation pipeline for conformation-specific antibodies should include:

• Biochemical assays with purified ORP3 in different ligand-bound states

• Structural validation using hydrogen-deuterium exchange mass spectrometry

• Cellular validation using mutant ORP3 locked in specific conformations

• Cross-validation using multiple detection methods (WB, IP, IF) under varying conditions

Strategic Development Timeline:

This development pipeline, while time-intensive, would yield valuable tools capable of distinguishing between cholesterol-bound, phosphoinositide-bound, and unbound states of ORP3, enabling unprecedented insights into the protein's dynamic functions in cells.