OPCML Antibody, HRP conjugated

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

OPCML is a GPI-anchored protein that functions as a tumor suppressor gene frequently silenced epigenetically in ovarian and other cancers. Its significance lies in its ability to suppress tumor growth by regulating a specific repertoire of receptor tyrosine kinases (RTKs), including EPHA2, FGFR1, FGFR3, HER2, and HER4 . The protein consists of three immunoglobulin-like domains and forms homodimers via contacts between membrane-distal domains . OPCML's frequent inactivation (in over 80% of ovarian cancer patients) through somatic methylation and loss of heterozygosity makes it a crucial target for cancer research .

What are the typical applications for HRP-conjugated OPCML antibodies?

HRP-conjugated OPCML antibodies are primarily used in:

• ELISA applications (recommended dilution 1:1000)

• Western blotting (recommended dilution 1:100-500)

• Detection of endogenous OPCML in human, rat, and mouse samples

• Investigating OPCML expression in normal vs. cancerous tissues

• Studying the correlation between OPCML expression and clinical outcomes in cancer patients

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experiments while maintaining sensitivity for detecting native and recombinant OPCML proteins .

How does OPCML antibody reactivity differ across species?

Current research demonstrates that anti-OPCML antibodies show variable cross-reactivity depending on the specific antibody clone and manufacturer. Based on the available data:

*Other predicted reactive species include pig, bovine, horse, sheep, rabbit, dog, and chicken, though these require validation for specific applications .

What are the optimal conditions for using HRP-conjugated OPCML antibodies in Western blot analysis?

For optimal Western blot results when using HRP-conjugated OPCML antibodies:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for tissue/cell lysis

• Protein loading: 20-40 μg of total protein per lane

• Separation: 10-12% SDS-PAGE gel recommended for optimal separation

• Transfer: Use PVDF membrane (0.45 μm pore size) with wet transfer (25V overnight at 4°C)

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute HRP-conjugated OPCML antibody 1:100-500 in blocking buffer and incubate overnight at 4°C

• Washing: 3-5 washes with TBST (5 minutes each)

• Direct development: Use ECL substrate for detection (no secondary antibody needed)

• Expected band: ~55-58 kDa for full-length OPCML

This methodology allows for specific detection of OPCML across human, rat, and mouse samples, with particular sensitivity for detecting the C-terminal region of the protein .

How can I optimize ELISA protocols when using HRP-conjugated OPCML antibodies?

For ELISA applications with HRP-conjugated OPCML antibodies:

• Coating: Immobilize capture antibody (non-conjugated anti-OPCML) at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C

• Blocking: 1-2% BSA in PBS for 1-2 hours at room temperature

• Sample: Apply diluted samples (cell lysates, serum, or recombinant protein) for 2 hours at room temperature

• Detection: Dilute HRP-conjugated OPCML antibody 1:1000 in blocking buffer and incubate for 1 hour at room temperature

• Washing: 4-5 washes with PBST between steps

• Substrate: TMB substrate solution for colorimetric detection

• Stop reaction: 2N H₂SO₄

• Readout: Measure absorbance at 450 nm with 570 nm reference

This protocol provides high sensitivity for detecting OPCML with a reported detection limit of approximately 10-50 pg/ml in research settings .

What controls should be included when validating OPCML antibody specificity?

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

• Positive tissue controls:

  • Cerebral cortex (high endogenous OPCML expression)

  • Normal ovarian surface epithelial cells (OSE-C2)

• Negative tissue controls:

  • OPCML-null cancer cell lines (SKOV3, PEO1)

  • Tissues from OPCML knockout models

• Experimental validation controls:

  • OPCML-overexpressing transfected cell lines (BKS-2.1)

  • OPCML knockdown lines using siRNA or shRNA

  • Peptide competition assay using the immunizing peptide

  • Secondary antibody-only control for IHC applications

• Cross-reactivity assessment:

  • Testing against other IgLON family members (LSAMP, NEGR1, HNT, IgLON5)

  • Pre-adsorption with recombinant OPCML protein

Proper validation ensures that the observed signals are specific to OPCML and not due to cross-reactivity or non-specific binding .

How can HRP-conjugated OPCML antibodies be used to investigate OPCML-RTK interactions?

To investigate OPCML-RTK interactions using HRP-conjugated OPCML antibodies:

• Co-immunoprecipitation:

  • Immunoprecipitate with antibodies against RTKs of interest (EPHA2, FGFR1, HER2)

  • Probe with HRP-conjugated OPCML antibody to detect interactions

  • Alternatively, immunoprecipitate with OPCML antibody and probe for RTKs

• Proximity Ligation Assay (PLA):

  • Use primary antibodies against OPCML and RTKs (e.g., AXL)

  • Apply PLA probes and ligase

  • Signal indicates protein proximity (<40 nm)

• Förster Resonance Energy Transfer (FRET):

  • Label OPCML and RTK with appropriate fluorophores

  • Measure energy transfer as indicator of molecular proximity

• Gas6-induced interaction studies:

  • Serum-starve cells expressing OPCML and AXL

  • Stimulate with Gas6 (AXL ligand) at various time points

  • Analyze co-localization and interaction using PLA or co-IP

These techniques have revealed that OPCML preferentially binds to activated RTKs, such as AXL after Gas6 stimulation, providing insight into the mechanism of OPCML's tumor suppressor function .

What approaches can be used to study OPCML's role in RTK trafficking and degradation?

To investigate OPCML's role in RTK trafficking and degradation:

• Subcellular fractionation:

  • Prepare detergent-soluble and detergent-insoluble (lipid raft) fractions

  • Use HRP-conjugated OPCML antibody to detect OPCML localization

  • Probe for RTKs to determine co-localization with OPCML in lipid rafts

• Ubiquitination assay:

  • Transfect cells with HA-tagged ubiquitin

  • Immunoprecipitate RTKs of interest (e.g., HER2)

  • Probe for ubiquitination using anti-HA antibody

  • Compare between OPCML-expressing and OPCML-null cells

• Proteasomal degradation pathway:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Analyze RTK levels in presence/absence of OPCML

  • Use HRP-conjugated OPCML antibody to confirm OPCML expression

• Non-clathrin mediated endocytosis:

  • Use endocytosis inhibitors specific to different pathways

  • Track RTK internalization and degradation

  • Analyze how OPCML affects trafficking routes

These methodologies have demonstrated that OPCML promotes RTK degradation through polyubiquitination and proteasomal mechanisms, particularly for EPHA2, FGFR1, and HER2, but not for unassociated RTKs like EGFR .

How can OPCML antibodies be used to investigate the relationship between OPCML expression and patient survival?

To investigate relationships between OPCML expression and patient survival:

• Tissue microarray analysis:

  • Use HRP-conjugated OPCML antibody for IHC staining of patient tissue microarrays

  • Score OPCML expression levels (negative, weak, moderate, strong)

  • Correlate with clinical data and survival outcomes

• OPCML/RTK ratio analysis:

  • Perform dual staining for OPCML and specific RTKs (e.g., AXL)

  • Calculate OPCML/AXL expression ratio for each patient

  • Stratify patients based on high vs. low OPCML/AXL ratio

  • Compare survival outcomes between groups

• Methylation-expression correlation:

  • Analyze OPCML promoter methylation status using methylation-specific PCR

  • Correlate with OPCML protein expression by IHC

  • Determine relationship with patient outcomes

How does OPCML expression affect RTK signaling pathways in cancer cells?

OPCML regulates RTK signaling through multiple mechanisms:

• Direct binding and sequestration:

  • OPCML binds to the extracellular domains of specific RTKs (EPHA2, FGFR1, FGFR3, HER2, HER4)

  • This interaction occurs preferentially when RTKs are activated by their ligands

  • For example, Gas6 stimulation enhances OPCML-AXL interaction

• Alteration of RTK localization:

  • OPCML resides in cholesterol-rich lipid domains (detergent-resistant membranes)

  • It recruits bound RTKs to these domains

  • This changes the microenvironment of RTKs and their signaling capabilities

• Promotion of RTK degradation:

  • OPCML facilitates polyubiquitination of bound RTKs

  • This leads to proteasomal degradation

  • Consequently reduces total RTK levels and signaling output

• Prevention of RTK cross-activation:

  • OPCML inhibits AXL-mediated transactivation of other RTKs (cMET and EGFR)

  • This prevents sustained phospho-ERK signaling

  • Suppresses induction of EMT transcription factors like Slug

These mechanisms collectively result in suppressed downstream signaling through pathways such as AKT, MAPK/ERK, and STAT3, ultimately inhibiting cell proliferation, migration, and invasion in cancer cells .

What role do OPCML mutations play in cancer development and progression?

OPCML mutations contribute to cancer development through various mechanisms:

• Structural and functional impacts:

  • Somatic missense mutations in OPCML have been identified across multiple cancer types

  • X-ray crystallography (2.65 Å resolution) revealed that OPCML consists of three immunoglobulin-like domains with specific homodimerization contacts

  • Mutations can disrupt protein structure, dimerization, or RTK-binding regions

• Phenotypic effects of mutations:

  • Alterations in anchorage-independent growth

  • Changed interactions with activated RTKs

  • Modified cellular migration and invasion capabilities

  • Enhanced tumor growth in vivo

• Clinical significance:

  • OPCML inactivation occurs in over 80% of ovarian cancer patients

  • While promoter hypermethylation is the predominant silencing mechanism, mutations provide an alternative inactivation route

  • The TCGA dataset shows loss of OPCML expression in 92% of high-grade serous ovarian cancers

• Prognostic implications:

  • High OPCML expression is a favorable prognostic factor for progression in ovarian cancer (HR=0.71, p=4.3e-05) and relapse in breast cancer (HR=0.57, p=2.2e-16)

  • OPCML mutations contributing to protein dysfunction may therefore worsen prognosis

These findings suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in various cancers by compromising its tumor suppressor functions .

How can OPCML antibodies be used to study the therapeutic potential of targeting OPCML pathways?

OPCML antibodies can facilitate therapeutic research through several approaches:

• Evaluating OPCML restoration strategies:

  • Use HRP-conjugated OPCML antibodies to confirm protein expression after gene therapy or demethylating treatment

  • Assess downstream effects on RTK levels, signaling, and cancer cell phenotypes

• Combinatorial therapy assessment:

  • Monitor how OPCML status affects sensitivity to RTK inhibitors

  • For example, OPCML expression enhances sensitivity to the AXL inhibitor R428 both in vitro and in vivo

  • The chick chorion allantoic membrane (CAM) assay showed approximately threefold enhanced sensitization to AXL inhibition in OPCML-expressing cells

• Development of recombinant OPCML therapy:

  • Use OPCML antibodies to track biodistribution and efficacy of recombinant OPCML domain 1-3 protein

  • This exogenous recombinant protein has shown inhibitory effects on EOC cell growth in vitro and in vivo

• Patient stratification markers:

  • OPCML expression status could inform treatment decisions

  • Patients with low OPCML/high RTK profiles might benefit more from specific RTK inhibitors

  • OPCML antibodies can help establish these expression profiles in patient samples

These research approaches highlight the potential of OPCML-based therapeutics for modulating RTK networks rather than targeting linear signaling systems, offering a novel strategy for cancer treatment .

What factors might affect the specificity and sensitivity of HRP-conjugated OPCML antibody assays?

Several factors can impact HRP-conjugated OPCML antibody performance:

• Epitope accessibility issues:

  • OPCML's localization in lipid rafts may require special detergents for extraction

  • Use of Triton X-100 or NP-40 (0.5-1%) with brief sonication improves extraction

  • GPI-anchored proteins like OPCML may require specific extraction methods for complete solubilization

• Cross-reactivity considerations:

  • OPCML belongs to the IgLON family, which includes similar proteins (LSAMP, NEGR1, HNT, IgLON5)

  • Antibodies targeting conserved regions may cross-react, especially in tissues expressing multiple IgLON members

  • Validation using knockout/knockdown controls is essential

• Post-translational modifications:

  • OPCML is N-glycosylated and GPI-anchored

  • These modifications may mask epitopes or affect antibody binding

  • Deglycosylation treatments may be necessary for consistent detection

• HRP conjugation effects:

  • Direct HRP conjugation can affect antibody avidity and epitope recognition

  • Batch-to-batch variation in conjugation efficiency

  • Regular quality control testing is recommended

• Tissue-specific expression patterns:

  • OPCML expression varies significantly across tissues

  • Cerebral cortex shows high expression, while many cancer cell lines show none

  • Appropriate positive and negative controls are crucial for each tissue type

Addressing these factors through careful experimental design and appropriate controls will improve the reliability of HRP-conjugated OPCML antibody assays in research applications.

How can researchers distinguish between different isoforms of OPCML using antibody-based methods?

To distinguish between OPCML isoforms using antibody-based methods:

• Isoform-specific epitope targeting:

  • Use antibodies that target unique regions of specific OPCML isoforms

  • Select antibodies raised against epitopes that differ between isoforms

  • Verify epitope specificity based on manufacturer's data

• Western blot analysis:

  • Different OPCML isoforms appear as distinct bands on Western blots

  • Major isoform produces a band at approximately 55-58 kDa

  • Alternative splice variants may yield additional bands

  • Use high-resolution SDS-PAGE (8-10%) for better separation of similar-sized isoforms

• PCR verification in parallel:

  • Complement antibody detection with RT-PCR using isoform-specific primers

  • Correlate protein detection with mRNA expression patterns

  • This multi-modal approach provides more reliable isoform identification

• Recombinant isoform controls:

  • Use recombinant OPCML isoforms as positive controls

  • Compare migration patterns with endogenous proteins

  • Perform peptide competition assays with isoform-specific peptides

• Mass spectrometry validation:

  • For definitive isoform identification, immunoprecipitate OPCML using the antibody

  • Analyze by mass spectrometry to identify specific peptides unique to each isoform

  • This approach provides unambiguous isoform determination

These strategies enable researchers to accurately identify and study specific OPCML isoforms, which may have distinct functions or tissue distributions relevant to cancer biology.

What are the key considerations when using HRP-conjugated OPCML antibodies in multiplex immunoassays?

When incorporating HRP-conjugated OPCML antibodies into multiplex immunoassays:

• Signal separation strategies:

  • HRP produces a broad chemiluminescent signal that may overlap with other detection systems

  • Consider using different enzyme conjugates (AP, β-gal) for other targets

  • Alternatively, employ sequential detection with HRP inactivation between steps

• Cross-reactivity prevention:

  • Test each antibody individually before multiplexing

  • Perform extensive blocking (5% BSA or commercial blocker) to minimize non-specific binding

  • Use isotype-specific secondary antibodies when multiple primary antibodies are from the same species

• Optimization for co-detection with RTKs:

  • OPCML interacts with specific RTKs (EPHA2, FGFR1, HER2)

  • This interaction may mask epitopes recognized by certain antibodies

  • Test antibody combinations to ensure no interference with detection

  • Consider fixation methods that preserve protein-protein interactions while maintaining epitope accessibility

• Quantification considerations:

  • HRP signal dynamics may differ from other detection systems

  • Establish standard curves for each target to ensure accurate quantification

  • Use appropriate software for unmixing overlapping signals in densitometric analysis

• Tissue-specific optimization:

  • Background autofluorescence/peroxidase activity varies between tissues

  • Include appropriate quenching steps (H₂O₂ treatment or commercial quenchers)

  • Optimize antibody concentrations for each tissue type to maximize signal-to-noise ratio

These considerations will help researchers develop robust multiplex assays incorporating HRP-conjugated OPCML antibodies alongside other detection reagents for comprehensive analysis of OPCML and its interacting partners.