ORP3B Antibody

What is ORP-3 and why is it significant for antibody-based research?

ORP-3, also known as Oxysterol-binding protein-related protein 3 (OSBPL3), is a member of the oxysterol-binding protein family with a molecular mass of approximately 100 kDa . It functions as a phosphoinositide-binding protein that associates with both cellular and endoplasmic reticulum (ER) membranes . Its significance stems from its role in multiple cellular processes, including ER-plasma membrane contact sites formation, regulation of RRAS signaling pathways, modulation of integrin β1 activation, and influence on cell adhesion and polarity . These diverse functions make ORP-3 antibodies valuable tools for investigating membrane contact sites, lipid transport, and cellular signaling networks.

What experimental applications are ORP-3 antibodies validated for?

According to available data, rabbit polyclonal ORP-3 antibodies have been validated for several key applications in research settings. These include immunohistochemistry on paraffin-embedded samples (IHC-P), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) . The validated reactivity is primarily with human samples, though potential cross-reactivity with other species may exist based on sequence homology . When selecting an ORP-3 antibody, researchers should verify that the specific epitope region (such as the aa 300-450 region in human OSBPL3) is appropriate for their experimental design and target detection needs.

What cellular compartments will ORP-3 antibodies typically label?

ORP-3 antibodies typically label both plasma membrane and endoplasmic reticulum compartments, reflecting the protein's dual localization and its role in membrane contact sites . In immunofluorescence experiments, researchers should expect to observe signals at the cell periphery (plasma membrane) as well as reticular patterns characteristic of ER distribution. Additionally, enrichment may be visible at regions where the ER comes into close proximity with the plasma membrane, representing membrane contact sites where ORP-3 functions to recruit VAPA to form ER-plasma membrane junctions . The specific pattern may vary depending on cell type and activation state of signaling pathways that influence ORP-3 localization.

How can ORP-3 antibodies be used to investigate membrane contact sites?

ORP-3 antibodies serve as valuable tools for analyzing ER-plasma membrane contact sites due to ORP-3's ability to bind to the ER membrane protein VAPA and recruit it to plasma membrane sites . To investigate these contacts, researchers should implement dual or triple immunofluorescence protocols combining the ORP-3 antibody with markers for ER (e.g., anti-VAPA, anti-calnexin) and plasma membrane (e.g., WGA, membrane phospholipid markers). Super-resolution microscopy techniques like STORM or STED are recommended for accurately resolving these closely apposed membranes (typically 10-30 nm apart). For functional studies, researchers can combine ORP-3 immunostaining with proximity ligation assays (PLA) to visualize ORP-3-VAPA interactions specifically at contact sites. Live-cell imaging experiments using tagged ORP-3 constructs followed by confirmatory antibody staining in fixed cells can provide dynamic information about contact site formation and stability.

What methodological approaches enable investigation of ORP-3's role in RRAS signaling using antibodies?

To study ORP-3's role in RRAS signaling and its effects on integrin β1 activation, researchers should implement a multi-faceted approach similar to methods used for analyzing other signaling complexes in cancer research . This approach includes:

• Co-immunoprecipitation experiments using ORP-3 antibodies followed by Western blotting for RRAS and VAPA to confirm complex formation

• Sequential immunoblotting for phosphorylated downstream effectors (similar to approaches described for ErbB pathway analysis )

• Flow cytometry with antibodies against active integrin β1 conformations after ORP-3 knockdown/overexpression

• Immunofluorescence analysis of adhesion structures using paxillin or vinculin as markers alongside ORP-3 staining

Signal quantification should follow methods similar to those described for analyzing ErbB signaling networks, with appropriate normalization controls . Experiments should include cells in both adherent and suspension states to capture the full spectrum of integrin activation states influenced by ORP-3-mediated signaling.

How can ORP-3 antibodies be utilized in lipid binding and transport studies?

ORP-3 exhibits binding preferences for specific phosphoinositides, particularly PI(3,4)P2 and PI(3,4,5)P3, as well as 25-hydroxycholesterol and cholesterol . To investigate these interactions using antibodies, researchers should employ a combination of techniques:

• Immunoprecipitation of ORP-3 followed by lipid extraction and mass spectrometry to identify bound lipids in different cellular contexts

• Immunofluorescence co-localization studies with specific phosphoinositide probes

• In vitro competition assays between ORP-3 antibodies and different lipid species to determine epitope-specific interference with lipid binding

For sterol transport assays, researchers can adapt methodology from other OSBP family studies, using fluorescent cholesterol analogs and tracking their movement in cells where ORP-3 function has been perturbed through antibody microinjection or membrane-permeable derivatives.

What are the optimal conditions for Western blotting with ORP-3 antibodies?

Based on protocols described for similar antibody applications in complex signaling pathway analysis , optimal Western blotting conditions for ORP-3 antibodies should include:

• Sample preparation: Lyse cells in buffer containing 0.05 M Hepes pH 7.7, 10% glycerol, 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, supplemented with protease and phosphatase inhibitors

• Protein loading: 100 μg of total protein per lane on 7-12% SDS-PAGE gels

• Transfer conditions: Cold temperature liquid transfer at constant voltage (100 V) for 2 hours onto PVDF membranes

• Blocking: PBS with 0.1% Tween-20 (PBS-T) supplemented with 5% skimmed milk at room temperature for 2 hours

• Primary antibody incubation: Dilute ORP-3 antibody in PBS-T with 3% BSA and 0.002% sodium azide, incubate at 4°C for 18 hours

• Secondary antibody: Use peroxidase-conjugated anti-rabbit IgG at 1:2,000 dilution in PBS-T for 1 hour at room temperature

• Detection: Enhanced chemiluminescence reagents with digital imaging systems for quantification

For ORP-3 specifically, expect bands at approximately 100 kDa, with validation of specificity through appropriate knockdown controls.

What immunofluorescence protocols yield optimal results with ORP-3 antibodies?

Optimal immunofluorescence protocols for ORP-3 detection should be adapted from approaches used for membrane-associated proteins in complex tissue architectures :

• Fixation: 10% neutral buffered formalin for 24 hours for tissue sections; 4% paraformaldehyde for 15 minutes for cultured cells

• Antigen retrieval: High pH buffer (pH 9.0) at 97°C for 20 minutes

• Antibody dilution: Typically 1:25 to 1:100 in blocking buffer, with optimization required for each lot

• Incubation: 1 hour at 37°C or overnight at 4°C

• Detection: Secondary antibody systems such as HQ amplification with fluorophore-conjugated tertiary reagents for enhanced sensitivity

• Analysis: Confocal or super-resolution microscopy to resolve membrane-associated structures

For co-localization studies, sequential antibody incubations with appropriate blocking steps between primary antibodies from different species will minimize cross-reactivity issues.

How should immunoprecipitation experiments be designed to study ORP-3 protein complexes?

To effectively immunoprecipitate ORP-3 and associated proteins, the experimental design should include:

• Crosslinking step: Optional but recommended for capturing transient interactions, using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate))

• Lysis conditions: Gentle lysis buffers containing 0.5-1% NP-40 or Triton X-100, with ionic strength adjusted to maintain specific interactions

• Pre-clearing: Incubation with protein A/G beads and non-immune IgG to reduce non-specific binding

• Antibody binding: Use 2-5 μg of ORP-3 antibody per mg of total protein

• Sequential elution strategies: To differentiate between direct and indirect binding partners

• Controls: Including IgG control immunoprecipitations and reciprocal co-IPs

For validating ORP-3-VAPA interactions specifically, reciprocal immunoprecipitation using both anti-ORP-3 and anti-VAPA antibodies is essential, with subsequent immunoblotting to confirm complex formation.

What are common issues with ORP-3 antibody specificity and how can they be addressed?

Common specificity issues with ORP-3 antibodies include cross-reactivity with other OSBP family members due to conserved domains. To address these issues:

• Validate antibody specificity using siRNA knockdown or CRISPR knockout controls

• Perform peptide competition assays using the immunizing peptide (e.g., amino acids 300-450 of human OSBPL3 )

• Compare staining patterns with multiple antibodies targeting different ORP-3 epitopes

• Test reactivity in tissues or cells known to have low or no ORP-3 expression

• For immunohistochemistry, include appropriate absorption controls

When analyzing data, researchers should be aware that certain antibody lots may recognize splice variants differently. Western blot analysis alongside immunostaining can help confirm that the detected signals correspond to the expected molecular weight of ORP-3 (approximately 100 kDa) .

How can conflicting data from different ORP-3 antibodies be reconciled?

When faced with conflicting data from different ORP-3 antibodies, researchers should implement a systematic reconciliation approach:

• Epitope mapping: Determine the exact binding regions of each antibody to identify if they recognize different domains or conformations of ORP-3

• Functional validation: Use complementary techniques like proximity ligation assays to confirm protein interactions detected by co-immunoprecipitation

• Cellular context analysis: Determine if differences reflect cell type-specific post-translational modifications or protein-protein interactions

• Quantitative analysis: Apply statistical methods similar to those used in antibody comparison studies to determine significance of observed differences

• Literature cross-reference: Compare findings with published data on ORP-3 localization and function

For publication, clearly report the specific antibody clone, lot number, and validation experiments performed to allow proper interpretation of potentially conflicting results.

What criteria should be used to evaluate ORP-3 antibody performance in different applications?

To evaluate ORP-3 antibody performance across applications, researchers should establish clear criteria:

• For Western blotting:

  • Band specificity (single band at expected molecular weight)

  • Signal-to-noise ratio (>10:1 preferred)

  • Linear dynamic range for quantification

  • Consistency across different sample types

• For immunofluorescence:

  • Expected subcellular localization pattern

  • Minimal background in negative control samples

  • Consistent staining across different fixation methods

  • Co-localization with known interaction partners (e.g., VAPA)

• For immunoprecipitation:

  • Efficiency of target capture (>50% depletion from lysate)

  • Minimal non-specific binding in control conditions

  • Ability to co-immunoprecipitate known interaction partners

Document performance metrics systematically to facilitate comparison across experiments and antibody lots, similar to approaches used in validation of therapeutic antibodies .

How do monoclonal versus polyclonal ORP-3 antibodies compare in research applications?

The choice between monoclonal and polyclonal ORP-3 antibodies depends on specific research requirements:

For initial characterization studies, polyclonal antibodies like the rabbit polyclonal described offer advantages in detection sensitivity. For highly specific mechanistic studies, especially those requiring consistent long-term results, monoclonal antibodies targeting functionally important domains would be preferable.

What advantages might bispecific antibody approaches offer for studying ORP-3 interactions?

Bispecific antibody approaches, similar to those described for ErbB receptor studies , could provide significant advantages for studying ORP-3 interactions:

• Simultaneous detection of ORP-3 and its binding partners (e.g., VAPA) in their native complex

• Enhanced sensitivity for detecting transient or low-abundance complexes

• Ability to modulate specific interactions while preserving others

• Potential for functional perturbation of specific binding interfaces

Using platforms like the BiXAb™ tetravalent format , researchers could generate bispecific antibodies targeting ORP-3 and VAPA or ORP-3 and phosphoinositide-binding domains. Such tools would enable not only detection but potentially manipulation of ORP-3 function in live cells, particularly for dissecting its dual roles in lipid transport and signaling complex assembly.

How do genetic approaches complement antibody-based methods for ORP-3 research?

An integrated approach combining antibody-based detection with genetic manipulation provides comprehensive insights into ORP-3 function:

For rigorous experimental design, researchers should combine antibody detection of endogenous ORP-3 with genetic approaches to confirm specificity and biological relevance of observed phenomena, following validation strategies similar to those used in therapeutic antibody development .