PCDH17 Antibody, FITC conjugated

What is PCDH17 and why is it an important research target?

Protocadherin 17 (PCDH17) is a member of the cadherin superfamily that functions as a potential tumor suppressor in various cancers. PCDH17 has gained significant research attention due to its frequent methylation and inactivation in several cancer types, including gastric and colorectal cancers, through molecular mechanisms such as deletion, mutation, and promoter methylation . The protein plays critical roles in modulating autophagy and apoptosis, with emerging evidence showing its involvement in chemosensitivity. Studies have demonstrated that PCDH17 can augment the sensitivity of colorectal cancer cells to chemotherapeutic agents like 5-fluorouracil (5-FU) by promoting JNK-dependent autophagic cell death, making it a valuable target for cancer research . Understanding PCDH17's function provides insights into cancer development mechanisms and potential therapeutic approaches.

How does FITC conjugation benefit PCDH17 antibody applications in research?

FITC (fluorescein isothiocyanate) conjugation provides significant advantages for visualizing and quantifying PCDH17 in research applications. This fluorescent tag emits green fluorescence (approximately 519 nm) when excited at 495 nm, enabling direct visualization of PCDH17 localization without requiring secondary antibody detection steps. The FITC-conjugated PCDH17 antibody allows for:

• Direct immunofluorescence microscopy for cellular localization studies

• Flow cytometry applications with simplified protocols

• High-sensitivity detection in fluorescence-based assays

• Multiplexing capabilities when combined with other fluorophore-conjugated antibodies targeting different wavelengths

• Time-efficient experimental protocols by eliminating secondary antibody incubation steps

This conjugation is particularly valuable when studying subcellular localization of PCDH17 in cancer cells or tracking expression changes following experimental manipulations such as drug treatments or genetic modifications .

What are the optimal protocols for using PCDH17-FITC antibody in flow cytometry experiments?

For optimal flow cytometry results with PCDH17-FITC antibody, researchers should follow this methodological approach:

Sample Preparation:

• Harvest cells (1-5×10^6) by centrifugation at 300×g for 5 minutes

• Wash twice with cold PBS containing 0.5% BSA

• For intracellular detection: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.1% Triton X-100 for 5 minutes

• For membrane proteins: Maintain cells in non-permeabilized state

Antibody Staining Protocol:

• Block non-specific binding with 2% normal serum from the same species as the secondary antibody for 30 minutes

• Incubate with PCDH17-FITC antibody (dilution 1:50-1:200, optimize for specific lot) for 30-60 minutes at 4°C in darkness

• Wash three times with PBS/0.5% BSA

• Resuspend cells in appropriate buffer for flow cytometry analysis

• Include appropriate isotype control (FITC-conjugated rabbit IgG) to establish gating strategy

Analysis Considerations:

• Use 488 nm laser for excitation and 530/30 nm bandpass filter for detection

• Compensate for spectral overlap if performing multicolor experiments

• Analyze at least 10,000 events per sample for statistical significance

• Include controls for autofluorescence and non-specific binding

This protocol has been successfully employed in studies of apoptosis detection in colorectal cancer cells treated with 5-FU, where PCDH17 expression significantly influenced treatment outcomes .

How can PCDH17-FITC antibodies be optimized for immunofluorescence microscopy?

Optimizing PCDH17-FITC antibodies for immunofluorescence microscopy requires attention to fixation methods, antibody concentration, and imaging parameters:

Fixation and Permeabilization Options:

Protocol Optimization:

• Grow cells on glass coverslips or chamber slides to 70-80% confluence

• Wash with PBS (3×)

• Fix with preferred method from table above

• Permeabilize with 0.1-0.5% Triton X-100 (5 min) if using paraformaldehyde

• Block with 1-5% normal serum or BSA (30-60 min)

• Incubate with PCDH17-FITC antibody at optimized dilution (typically 1:100-1:500) for 1-2 hours at room temperature or overnight at 4°C in darkness

• Wash with PBS (3× for 5 min each)

• Counterstain nuclei with DAPI (1 μg/ml, 5 min)

• Mount with anti-fade mounting medium

Advanced Considerations:

• Use titration experiments to determine optimal antibody concentration

• Include antigen retrieval steps (10 mM citrate buffer, pH 6.0, 95°C for 10-20 min) for formalin-fixed tissues

• Implement primary antibody omission controls to assess non-specific binding

• Consider photobleaching prevention by minimizing exposure time and using anti-fade reagents

• For co-localization studies, select complementary fluorophores with minimal spectral overlap

This approach has been successfully used to examine PCDH17 expression patterns in relation to autophagy markers like BECN1 in colorectal cancer tissues .

How does PCDH17 expression correlate with 5-FU sensitivity in colorectal cancer research?

Research has established a significant correlation between PCDH17 expression and 5-FU sensitivity in colorectal cancer that has important clinical implications. Immunohistochemistry studies of colorectal cancer tissues revealed that PCDH17 is more highly expressed in 5-FU-sensitive tumors compared to 5-FU-resistant cases . The correlation data shows:

Mechanistically, experimental evidence indicates that PCDH17 augments 5-FU sensitivity through multiple pathways:

• Promoting apoptosis via caspase-3 activation

• Inducing autophagic cell death through increased BECN1 expression and LC3B-II turnover

• Activating the JNK signaling pathway, which further enhances autophagic cell death

These findings suggest that PCDH17 expression status could potentially serve as a predictive biomarker for 5-FU response in colorectal cancer patients, possibly guiding treatment decisions in clinical settings .

What methodological approaches have been used to investigate PCDH17's role in autophagy regulation?

Researchers have employed multiple complementary approaches to elucidate PCDH17's role in autophagy regulation, building a comprehensive understanding of this tumor suppressor's function:

Expression Analysis Techniques:

• Immunohistochemistry (IHC): Used to detect PCDH17 and autophagy marker BECN1 in patient tissue samples, revealing co-expression patterns and clinical correlations .

• Western Blotting: Employed to quantify PCDH17 expression alongside autophagy markers (LC3B-II/I ratio, BECN1) and signaling molecules (phosphorylated JNK, c-Jun) .

Genetic Manipulation Strategies:

• Stable Transfection: pCMV6 Entry-PCDH17 plasmids were transfected into colorectal cancer cell lines (HCT116, SW480) using MegaTran 1.0 transfection reagent, followed by selection of stable clones expressing PCDH17 .

• RNA Interference: Short hairpin RNA (shRNA) was used to knockdown PCDH17 expression in PCDH17-transfected cells to confirm specificity of observed effects .

Autophagy Assessment Methods:

• LC3B-II Turnover Assay: Used to monitor autophagosome formation through Western blotting of LC3B-I conversion to LC3B-II .

• BECN1 Expression Analysis: Examined as a marker of autophagy induction, with demonstrated correlation to PCDH17 expression .

• Pharmacological Inhibitors: Autophagy inhibitors were employed to distinguish between autophagy-dependent and autophagy-independent mechanisms of cell death .

Signaling Pathway Analysis:

• JNK Pathway Inhibition: SP600125 (10 μM), a JNK inhibitor, was used to determine whether JNK activation is essential for PCDH17-induced autophagy .

• Phosphorylation Analysis: Western blotting for phosphorylated c-Jun assessed JNK pathway activation status .

Functional Outcome Measurements:

• Cell Viability Assays: CCK-8 assay to quantify cell viability after 5-FU treatment in the context of PCDH17 expression manipulation .

• Apoptosis Detection: Annexin V-FITC Apoptosis Detection Kit was used for flow cytometric analysis of apoptotic cells .

These methodological approaches collectively demonstrated that PCDH17 promotes autophagy through JNK pathway activation, and this autophagy plays a dominant role in PCDH17-induced cell death compared to apoptosis .

How can researchers effectively validate PCDH17-FITC antibody specificity for experimental applications?

Rigorous validation of PCDH17-FITC antibody specificity is essential for generating reliable experimental data. Researchers should implement a comprehensive validation strategy incorporating multiple complementary approaches:

1. Molecular Weight Verification:

• Perform Western blotting with positive and negative control lysates

• Confirm detection of a single band at the expected molecular weight for PCDH17 (~126 kDa)

• Include recombinant PCDH17 protein as a positive control

2. Epitope-Specific Controls:

• Pre-incubate antibody with recombinant PCDH17 protein (AA 18-243) to block specific binding sites

• Compare staining patterns between blocked and unblocked antibody samples

• Observe elimination of specific staining when blocking peptide is used

3. Genetic Manipulation Controls:

• Compare staining in PCDH17-overexpressing cells vs. vector controls

• Assess signal reduction in PCDH17 knockdown models using shRNA or siRNA

• Utilize CRISPR/Cas9-generated PCDH17 knockout cells as negative controls

4. Cross-Reactivity Assessment:

• Test antibody on cell lines from different species (the antibody is reported to be human-specific)

• Examine potential cross-reactivity with other protocadherin family members

• Include appropriate isotype controls (FITC-conjugated rabbit IgG)

5. Multiplatform Validation:

• Compare results across different detection methods (flow cytometry, immunofluorescence, ELISA)

• Confirm subcellular localization pattern is consistent with known PCDH17 biology

• Use alternative antibody clones targeting different PCDH17 epitopes as confirmatory tools

Advanced Validation Approaches:

• Mass spectrometry identification of immunoprecipitated proteins

• Parallel reaction monitoring (PRM) to quantify epitope-containing peptides

• Orthogonal validation using mRNA expression correlation with protein detection

Implementation of this comprehensive validation strategy ensures that experimental findings attributed to PCDH17 are genuinely reflective of its biology rather than artifacts of non-specific antibody binding .

What are the current limitations in PCDH17 research methodologies and how might they be addressed?

Current PCDH17 research faces several methodological limitations that researchers should consider when designing experiments and interpreting results:

1. Antibody Specificity and Cross-Reactivity Issues:

• Limitation: The polyclonal nature of available PCDH17 antibodies may introduce variability between lots and potential cross-reactivity with other protocadherin family members.

• Solution: Employ multiple antibodies targeting different epitopes for confirmation, perform rigorous validation as described in FAQ 4.1, and consider developing monoclonal antibodies for improved specificity.

2. In Vitro vs. In Vivo Translation Challenges:

• Limitation: Most PCDH17 functional studies rely on cell lines that may not fully recapitulate the tumor microenvironment influences on PCDH17 function.

• Solution: Develop organoid models that better reflect tissue architecture, utilize patient-derived xenografts, and complement in vitro findings with tissue analyses from patient cohorts.

3. Temporal Dynamics of PCDH17 Expression:

• Limitation: Current methodologies often capture static snapshots of PCDH17 expression rather than dynamic changes during disease progression or treatment.

• Solution: Implement time-course experiments, utilize inducible expression systems, and develop live-cell imaging approaches with fluorescently tagged PCDH17.

4. Mechanistic Ambiguity:

• Limitation: While PCDH17 has been linked to JNK-mediated autophagy, the precise molecular interactions and signaling pathway connections remain incompletely characterized.

• Solution: Apply proximity labeling techniques (BioID, APEX), conduct comprehensive interactome analyses, and use phosphoproteomics to identify direct substrates and signaling partners.

5. Clinical Translation Barriers:

• Limitation: The predictive value of PCDH17 as a biomarker for 5-FU sensitivity requires validation in larger, prospective clinical cohorts with standardized methodologies.

• Solution: Establish consensus IHC scoring systems, develop digital pathology algorithms for quantitative assessment, and conduct multi-center validation studies with standardized protocols.

6. Technical Challenges with FITC Conjugation:

• Limitation: FITC is susceptible to photobleaching and has pH sensitivity that may affect signal stability in certain experimental conditions.

• Solution: Consider alternative fluorophores (Alexa Fluor 488) with improved photostability, implement anti-fade strategies, and validate consistent performance across pH ranges relevant to experimental conditions.

Addressing these limitations through methodological innovations will significantly advance our understanding of PCDH17 biology and its potential clinical applications in cancer diagnostics and therapeutics .

What are common technical issues encountered when using PCDH17-FITC antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with PCDH17-FITC antibodies. Below are common issues and their systematic resolution strategies:

1. Weak or Absent Fluorescence Signal:

2. High Background or Non-specific Staining:

3. Inconsistent Staining Patterns:

4. Flow Cytometry-Specific Issues:

These troubleshooting approaches have proven effective in optimizing PCDH17-FITC antibody performance across various experimental contexts, including studies of its relationship with 5-FU sensitivity in colorectal cancer .

How can researchers design experiments to differentiate between PCDH17-mediated apoptosis and autophagy in cancer models?

Designing experiments to distinguish between PCDH17-mediated apoptosis and autophagy requires sophisticated methodological approaches that can delineate these distinct but interconnected cell death pathways:

Experimental Design Framework:

• Sequential Pathway Inhibition Strategy:

  • Apply specific inhibitors in parallel experimental groups:

    • Pan-caspase inhibitor Z-VAD-FMK (20-50 μM) to block apoptosis

    • Autophagy inhibitors (3-methyladenine 5 mM, chloroquine 50 μM, or Bafilomycin A1 100 nM)

    • Combination of both inhibitor types

  • Measure cell viability/death in PCDH17-expressing versus control cells under 5-FU treatment

  • Compare rescue effects to determine relative contribution of each pathway

• Genetic Manipulation Approach:

  • Generate multiple cell line variants:

    • PCDH17-overexpressing cells

    • PCDH17-overexpressing cells with BECN1 knockdown (autophagy deficient)

    • PCDH17-overexpressing cells with caspase-3 dominant negative (apoptosis deficient)

  • Assess 5-FU sensitivity in each variant to isolate pathway contributions

• Temporal Analysis of Pathway Activation:

  • Conduct time-course experiments (6, 12, 24, 48 hours post-treatment)

  • Monitor markers sequentially:

    • Early apoptosis: Annexin V binding, caspase-3/7 activation

    • Early autophagy: LC3B puncta formation, BECN1 upregulation

    • Late events: Membrane permeability, nuclear fragmentation

  • Determine which pathway activates first and whether inhibiting the primary pathway shifts cell death to the alternative mechanism

Specific Methodological Approaches:

Advanced Analytical Approaches:

• Live Cell Imaging:

  • Transfect cells with fluorescent reporters:

    • For apoptosis: FRET-based caspase sensors

    • For autophagy: GFP-LC3 for autophagosome visualization

  • Perform simultaneous imaging to track pathway activation in real-time

• Quantitative Systems Analysis:

  • Measure multiple parameters simultaneously (high-content imaging)

  • Apply mathematical modeling to determine the proportional contribution of each pathway

  • Utilize Bayesian network analysis to map pathway interdependencies

Research has demonstrated that autophagy plays a more dominant role in PCDH17-induced cell death than apoptosis, as autophagy inhibitors blocked cell death to a greater extent than the pancaspase inhibitor Z-VAD-FMK in PCDH17-expressing colorectal cancer cells treated with 5-FU . This type of comprehensive experimental approach enables precise delineation of the mechanistic contributions of each pathway to PCDH17's tumor suppressive functions.