OPCML Antibody, FITC conjugated

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

OPCML is a glycosylphosphatidylinositol (GPI)-anchored protein that localizes to the outer leaflet of the plasma membrane and functions as a tumor suppressor. It belongs to the IgLON superfamily of cell adhesion molecules. OPCML has gained significant attention in cancer research because it is frequently silenced epigenetically in over 80% of ovarian cancer patients, primarily through somatic methylation and loss of heterozygosity . When re-expressed in cancer cells, OPCML inhibits proliferation in vitro and tumorigenicity in vivo by binding and downregulating a specific subset of receptor tyrosine kinases (RTKs) . Recent studies have also demonstrated OPCML's antitumor effects in other cancers such as cholangiocarcinoma, where it suppresses cell proliferation by inducing apoptosis via AXL/STAT3 inactivation pathway .

What are the primary applications for FITC-conjugated OPCML antibodies?

FITC-conjugated OPCML antibodies are primarily utilized in:

• Immunofluorescence microscopy to detect OPCML expression in cultured cells and tissue sections

• Flow cytometry for quantitative analysis of OPCML expression in cell populations

• Monitoring changes in OPCML expression following experimental manipulations

• Investigating protein localization and trafficking within cells

• Co-localization studies with other proteins using multi-color fluorescence techniques

The antibodies have demonstrated effectiveness in detecting human, mouse, and rat OPCML , making them valuable tools for comparative studies across species.

How should FITC-conjugated antibodies be stored to maintain optimal activity?

FITC-conjugated antibodies, including OPCML antibodies, should be:

• Stored at 2-8°C in the dark to prevent photobleaching

• Never exposed to continuous light as this causes gradual loss of fluorescence

• Stored in the presence of a protein stabilizer and appropriate preservative (typically 0.01% sodium azide, though this requires caution as sodium azide yields highly toxic hydrazoic acid under acidic conditions)

• Aliquoted if frequent use is anticipated to minimize freeze-thaw cycles

• Kept in the original manufacturer's buffer unless specifically required otherwise

What is the recommended protocol for immunofluorescence staining using FITC-conjugated OPCML antibodies?

For optimal immunofluorescence staining with FITC-conjugated OPCML antibodies:

• Fix cells with an appropriate fixative (typically methanol or 4% paraformaldehyde)

• Permeabilize cells if intracellular staining is required

• Block with PBS containing 10% fetal bovine serum (FBS) for 20 minutes at room temperature to reduce non-specific binding

• Incubate with the FITC-conjugated OPCML antibody at a 1:500 dilution in PBS/10% FBS for 1 hour at room temperature in the dark

• Wash cells 2 × 5 minutes with PBS

• Observe cells with a fluorescence microscope equipped with a FITC filter

How can I validate the specificity of OPCML antibody binding in my experiments?

To validate OPCML antibody specificity:

• Include appropriate positive controls - cells or tissues known to express OPCML (e.g., normal ovarian epithelium)

• Include negative controls - cells with OPCML knockdown or tissues known not to express OPCML

• Perform blocking experiments with recombinant OPCML protein

• Compare staining patterns with multiple OPCML antibodies recognizing different epitopes

• Verify results with complementary techniques such as Western blotting or RT-PCR

• For FITC-conjugated antibodies specifically, include an isotype control conjugated to FITC at the same concentration as the antibody of interest

What considerations are important when designing co-staining experiments with FITC-conjugated OPCML antibodies?

When designing co-staining experiments:

• Consider spectral overlap - FITC has excitation/emission maxima at approximately 495/519 nm, potentially overlapping with other green fluorophores

• Select secondary fluorophores with minimal spectral overlap (e.g., TRITC, Cy5)

• Perform proper compensation when using flow cytometry to correct for spectral overlap

• Validate antibody combinations to ensure no cross-reactivity between primary or secondary antibodies

• Optimize antibody concentrations to achieve comparable signal intensities

• Consider sequential staining if cross-reactivity is an issue

• Include appropriate single-stained controls to verify specificity

How can FITC-conjugated OPCML antibodies be used to study methylation-induced silencing in cancer cells?

FITC-conjugated OPCML antibodies can be valuable tools in studying methylation-induced silencing:

• Quantify OPCML protein expression before and after treatment with demethylating agents (e.g., 5-aza-2'-deoxycytidine) using flow cytometry with FITC-conjugated OPCML antibodies

• Combine with methylation-specific PCR (MSP) to correlate protein expression with methylation status

• Perform immunofluorescence imaging to visualize cellular localization following demethylation treatment

• Investigate the effects of compounds like luteolin, which has been shown to decrease methylation of the OPCML promoter region and upregulate OPCML expression

• Develop high-throughput screening assays for compounds that reverse OPCML methylation using FITC-conjugated antibodies as readouts

Research has demonstrated that luteolin treatment reduces the methylation level of the OPCML promoter region and decreases global DNA methylation levels in breast cancer cells, as measured using LC-MS/MS . This approach could be extended to other cancer types where OPCML is epigenetically silenced.

What is the significance of OPCML mutations in cancer progression and how can FITC-conjugated antibodies help investigate these mechanisms?

OPCML mutations have significant implications for cancer progression:

• Somatic missense mutations in OPCML have been observed in various tumor types and can contribute to tumorigenesis

• FITC-conjugated OPCML antibodies can be used to:

  • Compare expression levels and localization patterns between wild-type and mutant OPCML

  • Investigate the impact of specific mutations on OPCML's interaction with receptor tyrosine kinases

  • Assess changes in cellular phenotypes (proliferation, migration, invasion) associated with different OPCML variants

  • Study the effect of mutations on OPCML dimerization, which occurs via contacts between membrane-distal domains

  • Evaluate the impact of mutations on OPCML's ability to induce apoptosis

Studies have shown that OPCML variants with representative clinical mutations demonstrate clear phenotypic effects, including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro, and tumor growth in vivo .

How does OPCML influence receptor tyrosine kinase signaling, and how can this be studied using FITC-conjugated antibodies?

OPCML's regulation of receptor tyrosine kinases (RTKs) can be investigated using FITC-conjugated antibodies through:

• Co-localization studies with FITC-OPCML antibodies and fluorescently labeled RTKs (EPHA2, FGFR1, FGFR3, HER2, and HER4)

• Live-cell imaging to monitor real-time interactions between OPCML and RTKs

• Flow cytometry to quantify changes in RTK expression levels following OPCML overexpression

• Immunoprecipitation followed by fluorescence detection to study physical interactions

• Analysis of downstream signaling pathway components (e.g., AXL/STAT3) following manipulation of OPCML expression

Research has shown that OPCML negatively regulates a specific RTK repertoire by binding to their extracellular domains, thus promoting RTK degradation via a polyubiquitination-associated proteasomal mechanism, ultimately leading to growth inhibition .

What is the relationship between OPCML expression and apoptosis in cancer cells?

The relationship between OPCML expression and apoptosis can be investigated using:

• Flow cytometry with Annexin V/propidium iodide and FITC-conjugated OPCML antibodies to simultaneously assess OPCML expression and apoptotic status

• Time-lapse fluorescence microscopy to monitor OPCML expression during apoptotic events

• Correlation analyses between OPCML expression and expression of pro-apoptotic and anti-apoptotic proteins

Research on cholangiocarcinoma has demonstrated that ectopic OPCML expression significantly reduced cell viability and increased apoptotic cell populations. In KKU-M213A cells, OPCML transfection increased the population of apoptotic cells from 8.4% to 14.2% (p<0.05) compared to control vector. Similarly, in KKU-100 cells, apoptotic cell population increased from 26.3% to 33.8% (p<0.01) . This suggests that OPCML inhibits proliferation of cancer cells by inducing cell apoptosis.

How can background fluorescence be reduced when using FITC-conjugated OPCML antibodies?

To minimize background fluorescence:

• Optimize blocking conditions - increase blocking time or use different blocking agents (BSA, normal serum, commercial blocking buffers)

• Titrate antibody concentration - test dilutions ranging from 1:50 to 1:500 to determine optimal signal-to-noise ratio

• Increase washing steps - add additional washes with PBS containing 0.1% Tween-20

• Use fresh fixatives and ensure complete fixation

• Include appropriate negative controls and isotype controls

• Pre-absorb antibodies with tissues or cells that show non-specific binding

• Use Sudan Black B (0.1-0.3%) to reduce autofluorescence, particularly for fixed tissues

• Consider using confocal microscopy to improve signal-to-noise ratio

What factors affect the detection sensitivity of OPCML using FITC-conjugated antibodies?

Several factors influence detection sensitivity:

• Antibody quality and epitope specificity - antibodies targeting different epitopes of OPCML may have varying sensitivities (e.g., antibodies targeting AA 28-322 versus C-terminal regions)

• Fixation method - different fixatives can affect epitope accessibility

• FITC photobleaching - minimize exposure to light during all steps

• Tissue or cell autofluorescence - particularly problematic in certain tissues

• Microscope settings - optimization of exposure time, gain, and offset parameters

• Antibody concentration - too low concentrations may miss low-expressing samples

• Signal amplification methods - consider tyramide signal amplification for low-abundance targets

• Tissue processing - overfixation can mask epitopes and reduce sensitivity

How should experiments be designed to study the relationship between OPCML methylation and protein expression using FITC-conjugated antibodies?

To effectively study the relationship between OPCML methylation and protein expression:

• Perform methylation-specific PCR or bisulfite sequencing to determine methylation status of the OPCML promoter

• Quantify OPCML protein expression using flow cytometry with FITC-conjugated antibodies

• Treat cells with demethylating agents (e.g., 5-aza-2'-deoxycytidine) and monitor changes in methylation and protein expression

• Include appropriate controls - cell lines with known methylation status

• Perform time-course experiments to determine the kinetics of demethylation and protein re-expression

• Consider using compounds like luteolin that have been shown to decrease methylation of the OPCML promoter region

What considerations are important when interpreting fluorescence intensity data from FITC-conjugated OPCML antibody experiments?

When interpreting fluorescence intensity data:

• Consider the relative stability of FITC fluorescence - FITC is more prone to photobleaching than some other fluorophores

• Account for potential autofluorescence, particularly in certain tissues or following certain treatments

• Use appropriate controls for normalization of fluorescence intensity

• Be aware that FITC intensity can be affected by pH changes in the microenvironment

• Consider quantitative approaches such as mean fluorescence intensity (MFI) calculation

• When using flow cytometry, ensure proper compensation for spectral overlap

• For time-course experiments, consider the potential degradation of fluorescence over time

• Remember that antibody binding may reach saturation, limiting the linear range of detection

By addressing these considerations, researchers can obtain more reliable and interpretable data from experiments using FITC-conjugated OPCML antibodies.

How can FITC-conjugated OPCML antibodies contribute to understanding the role of OPCML in other cancer types beyond ovarian cancer?

While OPCML has been extensively studied in ovarian cancer, FITC-conjugated antibodies can facilitate research in other cancer types:

• Perform immunofluorescence screening across tissue microarrays from multiple cancer types

• Correlate OPCML expression with clinical outcomes in various cancers

• Investigate cancer-specific interaction partners using co-localization studies

• Study the effects of cancer-specific mutations on OPCML function and localization

• Develop personalized medicine approaches based on OPCML status

Recent research has demonstrated OPCML's antitumor effects in cholangiocarcinoma, where it suppresses proliferation by inducing apoptosis via AXL/STAT3 inactivation and inhibits cell migration and invasion by downregulating Rho GTPases (RHOA, RAC1, and CDC42) . Studies have also shown that luteolin can affect OPCML methylation status in breast cancer cells , suggesting potential therapeutic applications across multiple cancer types.

What novel therapeutic approaches targeting OPCML are being investigated, and how can FITC-conjugated antibodies assist in these studies?

FITC-conjugated OPCML antibodies can support research into novel therapeutics by:

• Screening compounds that restore OPCML expression in methylated cancer cells

• Monitoring the efficacy of recombinant OPCML protein therapy

• Evaluating the effects of OPCML-targeted gene therapy approaches

• Studying combination therapies targeting OPCML and interacting RTKs

• Developing OPCML-based biomarkers for patient stratification