OPCML Antibody

What is OPCML and why is it important in cancer research?

OPCML (Opioid-binding protein/cell adhesion molecule-like) is a GPI-anchored protein that functions as a tumor suppressor gene. It belongs to the IgLON family of cell adhesion molecules, containing three immunoglobulin-like domains . OPCML is frequently silenced in various cancers, particularly ovarian cancer, through epigenetic mechanisms like DNA methylation and loss of heterozygosity (LOH) . Its importance stems from its role in regulating a specific repertoire of receptor tyrosine kinases (RTKs), including EPHA2, FGFR1, FGFR3, HER2, and HER4, by binding to their extracellular domains and promoting their degradation, thereby inhibiting cancer cell growth .

What are the common applications for OPCML antibodies in research?

OPCML antibodies are primarily used for:

• Western blotting (WB) to detect protein expression levels

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) to examine tissue localization

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

• Immunofluorescence microscopy to study subcellular localization and co-localization with other proteins

• Co-immunoprecipitation to investigate protein-protein interactions

• Proximity ligation assays (PLA) to detect protein interactions in situ

What types of OPCML antibodies are available for research?

According to the search results, researchers can access both:

• Polyclonal antibodies: Including rabbit polyclonal antibodies that recognize human, mouse, and rat OPCML

• Monoclonal antibodies: Such as rabbit recombinant monoclonal antibodies and mouse monoclonal antibodies

The antibodies are available in different formats, predominantly as unconjugated primary antibodies for various detection methods .

How should OPCML antibodies be stored and handled for optimal performance?

OPCML antibodies should be stored at -20°C and are typically stable for one year after shipment . For reconstituted antibodies, they can be stored at 2-8°C for approximately one month under sterile conditions or at -20°C to -70°C for six months . The antibodies are generally supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is often unnecessary for -20°C storage , but it's recommended to avoid repeated freeze-thaw cycles to maintain antibody performance .

What dilutions are recommended for different applications of OPCML antibodies?

Based on the search results, the following dilutions are recommended:

It's important to note that optimal dilutions should be determined by each laboratory for each specific application and sample type, as mentioned in the product information .

How can I validate the specificity of an OPCML antibody for my experimental system?

To validate OPCML antibody specificity:

• Positive and negative controls:

  • Use tissues or cell lines known to express OPCML (e.g., brain tissue for high expression , ovarian cancer cell lines with confirmed OPCML expression)

  • Include OPCML-null samples (e.g., cancer cell lines with confirmed methylation silencing of OPCML like SKOV3-Empty )

• Molecular weight verification:

  • OPCML is a glycosylated protein with a predicted molecular weight of 38 kDa

  • The observed molecular weight is typically around 45-50 kDa due to glycosylation

• Knockdown/overexpression systems:

  • Use stable OPCML knockdown (KD) cell lines created with shRNA or siRNA

  • Compare with cells ectopically expressing OPCML through transfection

• Biological function validation:

  • Confirm that antibody-detected OPCML correlates with expected biological functions (e.g., decreased RTK signaling, reduced cell proliferation)

How can OPCML antibodies be used to study protein-protein interactions with RTKs?

OPCML antibodies can be effectively used to study protein-protein interactions with RTKs through several methods:

• Co-immunoprecipitation (Co-IP):

  • Use anti-OPCML antibodies to pull down OPCML complexes and detect associated RTKs (like AXL, HER2, EPHA2) by western blotting

  • Reciprocal Co-IP using RTK antibodies (e.g., anti-AXL) can confirm interactions by detecting OPCML in the pulled-down complex

• Proximity Ligation Assay (PLA):

  • PLA can detect protein proximity (<40 nm) in situ, allowing visualization of OPCML-RTK interactions within their cellular context

  • This has been successfully used to demonstrate Gas6-induced interactions between OPCML and AXL in cancer cells

• Förster Resonance Energy Transfer (FRET):

  • FRET has been used to confirm the proximity between OPCML and AXL, providing evidence of direct molecular interaction

• Mammalian 2-hybrid assay:

  • This technique has been employed to detect interactions between OPCML and the extracellular domain of AXL cloned in a pVP16 vector

• GST pull-down assays:

  • GST-tagged recombinant OPCML proteins can pull down RTKs from cell lysates, confirming direct binding

  • This approach helped establish that OPCML interacts with specific RTKs through their extracellular domains

What are the best methods to study OPCML-mediated RTK degradation using antibodies?

To study OPCML-mediated RTK degradation:

• Comparative western blotting:

  • Compare RTK levels (total and phosphorylated) in OPCML-expressing versus OPCML-null cells

  • Monitor RTK degradation kinetics following ligand stimulation (e.g., Gas6 for AXL, EGF for EGFR family)

• Proteasome inhibition experiments:

  • Use proteasome inhibitors alongside OPCML antibodies to determine if the degradation mechanism is proteasome-dependent

  • Western blotting can reveal accumulated ubiquitinated RTKs when proteasomes are inhibited in OPCML-expressing cells

• Polyubiquitination detection:

  • Use antibodies against ubiquitin alongside OPCML and RTK antibodies to detect polyubiquitinated RTK species

  • This helps confirm the "polyubiquitination-associated proteasomal mechanism" of OPCML-mediated RTK degradation

• Pulse-chase experiments:

  • These can determine the half-life of RTKs in the presence or absence of OPCML

  • OPCML antibodies can be used to confirm expression during these experiments

• Co-localization with trafficking markers:

  • Use OPCML antibodies alongside markers for different endocytic compartments to track altered trafficking of RTKs

  • This helps understand the "non-clathrin dependent endocytosis" mechanism described in the literature

How can I use OPCML antibodies to study its role as a tumor suppressor in different cancer models?

To study OPCML's tumor suppressor function using antibodies:

How do I address discrepancies in observed molecular weight when detecting OPCML?

When addressing molecular weight discrepancies in OPCML detection:

What factors affect OPCML antibody detection in cancer tissues with epigenetic silencing?

Several factors can affect OPCML antibody detection in cancer tissues with epigenetic silencing:

• Methylation heterogeneity:

  • OPCML is frequently silenced by promoter methylation in various cancers

  • Methylation patterns can be heterogeneous within a tumor, leading to variable OPCML expression

  • Solution: Consider using methylation-specific PCR alongside antibody detection to correlate methylation status with protein expression

• Antibody sensitivity limitations:

  • Low-level expression may be below detection threshold for some antibodies

  • Solution: Use more sensitive detection methods (e.g., amplified detection systems, highly validated antibodies)

• Technical considerations:

  • Antigen retrieval methods are critical for IHC in fixed tissues, especially for GPI-anchored proteins

  • Solution: Optimize antigen retrieval protocols specifically for OPCML detection

• Biological considerations:

  • Other mechanisms beyond methylation (e.g., LOH) can lead to OPCML loss

  • Solution: Complement protein detection with genetic analysis (e.g., LOH assessment)

• Expression reactivation:

  • Treatment with demethylating agents can reactivate OPCML expression

  • Solution: Consider using such agents as positive controls in experimental models

How can I optimize co-immunoprecipitation protocols for detecting OPCML interactions with RTKs?

To optimize co-immunoprecipitation (Co-IP) for OPCML-RTK interactions:

• Lysis buffer optimization:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Consider specialized buffers for membrane proteins that contain GPI anchors

  • Include appropriate protease and phosphatase inhibitors to prevent degradation

• Pre-clearing strategy:

  • Pre-clear lysates with control agarose resin to reduce non-specific binding

  • This is especially important when working with cancer cell lines that may have high background

• Antibody selection and immobilization:

  • For OPCML pull-down, use antibodies targeting epitopes away from RTK binding sites

  • For RTK pull-down (e.g., AXL), consider both total and phospho-specific antibodies

  • Proper antibody immobilization is critical; methods using AminoLink Plus Coupling Resin have been successful

• Stimulation conditions:

  • Some interactions are enhanced by ligand stimulation (e.g., Gas6 for AXL-OPCML interaction)

  • Consider serum starvation followed by specific ligand treatment to enhance detectable interactions

• Controls and validation:

  • Include appropriate negative controls (isotype antibodies, OPCML-negative cells)

  • Validate findings with reciprocal Co-IP (pull down with RTK antibody, detect OPCML)

  • Confirm findings with complementary methods (PLA, FRET, GST pull-down)

How can OPCML antibodies be used to evaluate the potential of recombinant OPCML as a therapeutic agent?

OPCML antibodies play a crucial role in evaluating recombinant OPCML (rOPCML) as a potential therapeutic agent:

• Verification of rOPCML integrity and activity:

  • Use antibodies to confirm the structural integrity of rOPCML in stability studies

  • Verify that the recombinant protein maintains its ability to bind target RTKs through Co-IP or other binding assays

• Biodistribution studies:

  • Track rOPCML localization in preclinical models using antibody-based detection methods

  • Determine whether rOPCML effectively reaches tumor tissues following administration

• Mechanism of action studies:

  • Use antibodies to confirm that rOPCML recapitulates the normal functions of endogenous OPCML:

    • RTK binding and downregulation

    • Inhibition of downstream signaling pathways (e.g., AKT, ERK)

    • Anti-proliferative effects

• Monitoring treatment efficacy:

  • In preclinical models, use antibodies to assess changes in target RTK levels and phosphorylation status

  • Correlate these molecular changes with phenotypic outcomes (tumor regression, reduced proliferation)

• Companion diagnostics development:

  • OPCML antibodies could be developed into companion diagnostics to identify patients likely to benefit from rOPCML therapy

  • Patients with tumors showing low OPCML expression but high expression of OPCML-regulated RTKs may benefit most

• Combination therapy assessment:

  • Evaluate the effects of combining rOPCML with current standard therapies using antibody-based readouts

  • Assess whether rOPCML enhances the efficacy of chemotherapy or targeted therapies by modulating RTK networks

What methodologies can be used to study the effect of OPCML mutations on its tumor suppressor function?

To study the effect of OPCML mutations on its tumor suppressor function:

• Structural-functional analysis:

  • Use antibodies to verify expression of OPCML mutants in experimental systems

  • The X-ray crystal structure of OPCML (2.65 Å resolution) provides a framework for understanding mutation effects

  • Focus on mutations affecting the extended arrangement of three immunoglobulin-like domains or homodimerization interfaces

• Mutation expression systems:

  • Generate a panel of OPCML variants with clinically relevant mutations

  • Use site-directed mutagenesis to introduce specific mutations into OPCML expression constructs

  • Transfect these constructs into OPCML-null cell lines and use antibodies to confirm expression

• Functional assays:

  • Compare wild-type and mutant OPCML effects on:

    • Anchorage-independent growth

    • Interaction with activated RTKs (using Co-IP with OPCML antibodies)

    • Cellular migration and invasion

    • Tumor growth in vivo

• Protein-protein interaction studies:

  • Use methods like mammalian 2-hybrid assays, GST pull-downs, and Co-IP with OPCML antibodies

  • Compare RTK binding profiles of wild-type versus mutant OPCML

  • Determine if mutations affect OPCML homodimerization

• Localization studies:

  • Use immunofluorescence with OPCML antibodies to determine if mutations affect:

    • Cell surface localization

    • Lipid raft association

    • Co-localization with target RTKs

How can OPCML antibodies be used to investigate the epigenetic reactivation of OPCML expression?

OPCML antibodies are essential tools for investigating epigenetic reactivation of OPCML expression:

• Demethylating agent studies:

  • Use OPCML antibodies to monitor protein expression following treatment with DNA methyltransferase inhibitors (e.g., 5-azacytidine)

  • Correlate changes in protein expression with changes in methylation status assessed by methylation-specific PCR

• Dietary compound studies:

  • Natural compounds like luteolin have been shown to upregulate OPCML expression in breast cancer cells by reducing methylation

  • OPCML antibodies can confirm protein upregulation following such treatments

• Methylation activity assessment:

  • Combine OPCML antibody-based protein detection with assays measuring:

    • Global DNA methylation levels

    • OPCML promoter-specific methylation

    • DNA methyltransferase activity

• Functional consequences:

  • After epigenetic reactivation, use OPCML antibodies to:

    • Confirm increased protein expression

    • Assess effects on downstream RTK signaling

    • Monitor changes in cell behavior (proliferation, migration, etc.)

• Combination therapy approaches:

  • OPCML antibodies can help evaluate the efficacy of combining epigenetic modulators with RTK inhibitors

  • For example, assess whether demethylating agents that reactivate OPCML expression sensitize cells to AXL inhibitors like R428

• Clinical correlation:

  • In patient samples, correlate OPCML protein expression (detected by antibodies) with methylation status

  • This approach has been used to establish that OPCML methylation is a significant risk factor in ovarian cancer