Met Antibody, FITC conjugated

What is c-Met and why are FITC-conjugated c-Met antibodies important research tools?

c-Met (also known as MET or HGFR) is a 145 kDa receptor tyrosine kinase (RTK) expressed by epithelial cells of the brain, kidney, liver, and other tissues. It plays crucial roles in cell motility, angiogenesis, morphogenesis, proliferation, survival, and tissue regeneration . The importance of FITC-conjugated c-Met antibodies stems from c-Met's significant role in cancer biology, where its aberrant activation contributes to tumor development, invasion, metastasis, and drug resistance .

FITC-conjugated c-Met antibodies enable researchers to:

• Detect and quantify c-Met expression on cell surfaces via flow cytometry

• Visualize c-Met localization through immunofluorescence microscopy

• Monitor changes in c-Met expression following experimental interventions

• Study c-Met in its native conformation on live cells

These antibodies are particularly valuable for studying the complex signaling mechanisms triggered by c-Met activation, which involve multiple downstream effectors including PI3-kinase, PLCG1, SRC, GRB2, and STAT3, leading to activation of RAS-ERK, PI3K-AKT, and PLCγ-PKC signaling cascades .

How do researchers distinguish between different types of c-Met antibodies and their specific applications?

c-Met antibodies can be categorized based on several characteristics that determine their research utility:

Researchers should select antibodies based on:

• The specific c-Met domain being studied (e.g., extracellular vs. intracellular)

• The detection method being employed (flow cytometry, Western blot, immunoprecipitation)

• Whether native or denatured c-Met needs to be recognized

• The experimental temperature conditions to be used

For example, the seeMet 2 antibody shows strong binding to native c-Met in flow cytometry but is temperature-sensitive with reduced binding at 4°C compared to 37°C .

What are the optimal protocols for flow cytometric analysis using c-Met FITC antibodies?

A robust flow cytometry protocol for c-Met FITC antibodies typically follows these methodological steps:

• Cell Preparation:

  • Harvest cells (typically 1×10^5 to 1×10^8 cells per test)

  • Wash cells twice with PBS or TBS (pH 7.4)

  • Resuspend cells at appropriate concentration (typically 1×10^6 cells/100μL)

• Staining Procedure:

  • For cell surface c-Met: Incubate live cells with antibody

  • For total c-Met: Fix and permeabilize cells before antibody incubation

  • Optimal antibody concentration: 1-10 μg/mL (e.g., 5 μL of a 0.5 mg/mL solution)

  • Incubation time: 30-60 minutes at appropriate temperature (note temperature sensitivity of certain clones)

  • Wash cells 2-3 times with buffer containing 0.1-0.5% BSA

• Critical Controls:

  • Isotype control antibody conjugated to FITC

  • Unstained cells for autofluorescence baseline

  • Positive control (cell line with known high c-Met expression, e.g., SNU-5 or HepG2)

  • Negative control (cell line with low c-Met expression, e.g., T47D)

• Instrument Settings:

  • Excitation: 488 nm (blue laser)

  • Emission detection: 520 nm

  • Proper compensation if using multiple fluorophores

• Analysis Considerations:

  • Gate on viable cells (using appropriate viability dye)

  • Analyze difference in median fluorescence intensity between sample and controls

  • Quantify percentage of c-Met positive cells based on appropriate gating strategy

For researchers investigating temperature-sensitive antibodies like seeMet 2, parallel sample preparation at both 4°C and 37°C is recommended to capture the full spectrum of binding characteristics .

How should researchers prepare samples for immunofluorescence microscopy with c-Met FITC antibodies?

Optimal immunofluorescence sample preparation for c-Met FITC antibodies requires attention to several critical parameters:

• Sample Preparation and Fixation:

  • For adherent cells: Culture on glass coverslips or chamber slides

  • Wash cells twice with TBS to remove media components

  • Fix cells with freshly prepared methanol:acetone (1:1) mixture for 1 minute at room temperature

  • Alternative fixation: 4% paraformaldehyde for 10-15 minutes (preserves membrane structures)

  • Wash fixed cells with TBS four times

• Permeabilization (for intracellular epitopes):

  • If using paraformaldehyde fixation, permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  • Wash three times with TBS

• Blocking and Antibody Incubation:

  • Block with 10% normal serum (e.g., goat serum) to reduce non-specific binding

  • Incubate with c-Met-FITC antibody at 1-10 μg/mL in TBS at room temperature for 1 hour

  • For temperature-sensitive antibodies, maintain appropriate temperature throughout

  • Wash cells with TBS twice

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI or Hoechst dye

  • Mount using anti-fade mounting medium containing anti-photobleaching agents:

    • Propyl gallate

    • p-phenylenediamine dihydrochloride

    • 1,4-Diazabicyclo[2.2.2]octane (DABCO)

• Microscopy Settings:

  • Use appropriate filter set for FITC visualization:

    • Excitation maximum: 492 nm

    • Emission maximum: 520 nm

  • Minimize exposure time to reduce photobleaching

  • Capture images promptly after preparation

Troubleshooting guidance for common immunofluorescence issues:

How can temperature-sensitive c-Met antibodies be leveraged for studying receptor conformation and dynamics?

Temperature-sensitive c-Met antibodies like seeMet 2 present unique opportunities for investigating c-Met receptor dynamics:

The temperature sensitivity of seeMet 2, characterized by strong binding at 37°C but significantly reduced binding at 4°C, suggests recognition of a cryptic epitope within the c-Met α-chain that becomes accessible only at physiological temperatures . This property can be strategically utilized to:

• Study Receptor Conformational Changes:

  • Comparing binding patterns at different temperatures (4°C vs. 37°C) to map conformational transitions

  • Investigating how ligand binding or drug interactions affect epitope accessibility

  • Analyzing domains that undergo temperature-dependent structural rearrangements

• Investigate Membrane Fluidity Effects:

  • Correlating temperature-dependent binding with membrane lipid phase transitions

  • Studying how membrane microdomains influence c-Met accessibility and function

  • Examining receptor clustering behavior at different temperatures

• Research Protocol Design:

  • Pre-incubate cells at variable temperatures (4°C, 25°C, 37°C) before antibody addition

  • Perform parallel flow cytometry analyses at multiple temperatures

  • Use confocal microscopy with temperature-controlled stage to visualize dynamic changes

  • Incorporate lipid-altering agents (e.g., cholesterol depletion) to assess membrane effects

• Data Analysis Approach:

  • Quantify the temperature-dependent binding coefficient (ratio of binding at 37°C vs. 4°C)

  • Create Arrhenius plots to determine activation energy of conformational transitions

  • Use computational modeling to predict structural changes underlying temperature sensitivity

This temperature sensitivity might reflect physiologically relevant conformational states that could be exploited for targeted therapeutic interventions or as biomarkers for specific c-Met activation states in cancer .

How do researchers distinguish between agonistic and non-agonistic c-Met antibodies, and why is this critical for cancer research?

Distinguishing between agonistic and non-agonistic c-Met antibodies is crucial in cancer research due to the paradoxical effects of c-Met activation:

Significance in Cancer Research:
Many antibodies against c-Met unintentionally activate the receptor, potentially promoting rather than inhibiting cancer cell growth. This agonistic activity presents a major challenge in developing therapeutic antibodies against c-Met .

Methodological Approaches for Characterization:

• Mechanism-Based Dual Screening Assay:

  • Step 1: Assess Akt phosphorylation in appropriate cell lines (e.g., Caki-1 renal carcinoma cells)

  • Step 2: Determine c-Met degradation in cancer cells overexpressing c-Met (e.g., MKN45)

  • Goal: Identify antibodies that induce c-Met degradation while minimizing Akt activation

• Molecular and Cellular Analysis:

  • Immunoblot with phospho-specific antibodies to detect c-Met phosphorylation (pY1234/1235)

  • Measure downstream signaling activation (pAkt, pERK)

  • Assess cell scatter/migration assays to determine functional outcomes

  • Evaluate cell proliferation and survival in c-Met-dependent cell lines

• Comprehensive Characterization Framework:

• Examples from Research:

  • The antibody F46 demonstrates high affinity to human c-Met (Kd = 2.56 nM) with minimal agonistic activity while inducing c-Met degradation

  • Antibody 5D5 shows high agonistic activity despite high binding affinity

  • P3D12 antibody induces c-Met degradation with minimal activation of c-Met signaling

• Advanced Research Applications:

  • Development of biparatopic antibodies that bind two different epitopes simultaneously

  • Creation of antibody-drug conjugates (ADCs) using non-agonistic antibodies

  • Engineering of antibodies that preferentially target cancer-specific c-Met conformations

Non-agonistic c-Met antibodies with degradation-inducing properties represent valuable tools for both basic research and potential therapeutic development against c-Met-driven cancers .

What methodological approaches can maximize signal-to-noise ratio when using FITC-conjugated c-Met antibodies?

Optimizing signal-to-noise ratio is critical for generating reliable data with FITC-conjugated c-Met antibodies:

• Optimal Conjugation Parameters:

  • The fluorescein/protein (F/P) ratio significantly impacts performance

  • Optimal conjugation occurs at pH 9.5, room temperature, with protein concentration ~25 mg/ml

  • Reaction time of 30-60 minutes achieves maximal labeling

  • Separation of optimally labeled antibodies from under/over-labeled proteins using gradient DEAE Sephadex chromatography

• Minimizing Autofluorescence and Background:

  • Cell fixation: Use methanol:acetone (1:1) mixture rather than formaldehyde when possible

  • Block with 10% serum from species unrelated to the antibody source

  • Include 0.1-0.5% BSA in all wash and incubation buffers

  • Consider spectral unmixing for tissues with high autofluorescence

  • For cell sorting applications, maintain cells at appropriate temperature for antibody binding

• pH Considerations for FITC Signal Optimization:

  • FITC fluorescence is pH-dependent with optimal intensity at pH 8-9

  • Standardize buffer pH for consistent results across experiments

  • Consider using phosphate buffer (pH 7.4) supplemented with 5-10 mM sodium bicarbonate for imaging applications

• Advanced Sample Processing Techniques:

  • For tissue sections: Consider antigen retrieval methods optimized for c-Met

  • For cells with variable c-Met expression: Use brighter fluorophores (e.g., CF488A) for low-expressing samples

  • For multiplexed imaging: Ensure proper compensation to account for spectral overlap

• Signal Amplification Methods:

  • Tyramide signal amplification for low abundance targets

  • Multi-layer detection systems (using biotinylated secondary + streptavidin-FITC)

  • Avoid multiple amplification steps for quantitative applications

• Recommended Controls and Validation:

  • Negative control samples (known c-Met negative cells/tissues)

  • Blocking controls (pre-incubation with unlabeled antibody)

  • Absorption controls (pre-incubation of antibody with recombinant c-Met)

  • Titration experiments to determine optimal antibody concentration

  • Parallel validation with alternative detection methods (e.g., Western blot)

Implementing these methodological approaches enables researchers to achieve optimal signal-to-noise ratios, particularly important for detecting subtle changes in c-Met expression or localization in both normal and pathological samples.

How can c-Met FITC antibodies be used to study resistance mechanisms to c-Met tyrosine kinase inhibitors?

c-Met FITC antibodies serve as valuable tools for investigating resistance mechanisms to c-Met tyrosine kinase inhibitors (TKIs), providing insights at both cellular and molecular levels:

This approach not only helps understand resistance mechanisms but also guides the development of next-generation therapeutics, such as c-Met antibody-drug conjugates that maintain efficacy in TKI-resistant settings .

What strategies can researchers employ when using FITC-conjugated c-Met antibodies to study receptor heterogeneity in tumor samples?

Tumor heterogeneity presents significant challenges for cancer therapy, and FITC-conjugated c-Met antibodies provide powerful tools to investigate this complexity:

• Multi-Parameter Flow Cytometry Approaches:

  • Design panels combining c-Met-FITC with markers of:

    • Cancer stem cells (CD44, CD133)

    • Epithelial-mesenchymal transition (E-cadherin, Vimentin)

    • Co-expressed RTKs (EGFR, RON, HER2)

  • Implement index sorting to correlate c-Met expression with functional assays

  • Use high-dimensional analysis (tSNE, UMAP) to identify distinct cellular subpopulations

• Spatial Heterogeneity Analysis:

  • Apply immunofluorescence microscopy to map c-Met distribution within tumor sections

  • Perform digital image analysis to quantify regional variation

  • Implement tissue cytometry approaches combining the advantages of flow cytometry and histology

  • Correlate with tumor microenvironment features (hypoxia, immune infiltration)

• Addressing Co-Expression with Related Receptors:
  Research has shown that c-Met often exhibits heterogeneous co-expression with other RTKs:

  • c-Met and RON show differential expression patterns in >40% of pancreatic and triple-negative breast cancers

  • Develop dual-staining protocols to simultaneously detect c-Met and partner receptors

  • Use spectral unmixing to resolve fluorophore overlap in multiplexed imaging

• Methodological Considerations for Heterogeneity Studies:

  • Single-cell suspensions for flow cytometry:

    • Gentle enzymatic digestion to preserve surface epitopes

    • Immediate analysis or proper fixation to maintain antigen integrity

  • Tissue section analysis:

    • Serial sectioning to capture 3D heterogeneity

    • Automated whole-slide scanning for comprehensive spatial assessment

    • Registration with other markers or imaging modalities (H&E, IHC)

• Advanced Applications:

  • Patient-derived xenograft (PDX) models to maintain tumor heterogeneity

  • Single-cell RNA-seq correlation with c-Met protein expression

  • Cell sorting based on c-Met expression for downstream functional assays

  • Monitoring changes in heterogeneity during treatment response and resistance development

• Analytical Framework for Heterogeneity Quantification:

  Heterogeneity Aspect	Analytical Approach	Metrics
  Expression Level	Flow cytometry	Coefficient of variation, % positive cells
  Spatial Distribution	Immunofluorescence	Moran's I, Ripley's K function
  Co-expression Patterns	Multiplexed imaging	Pearson's correlation, overlap coefficient
  Functional Impact	Sorted cell assays	Differential drug sensitivity, growth rates

Understanding c-Met heterogeneity through these approaches can inform the development of more effective targeted therapies, including bispecific antibodies or dual-targeting antibody-drug conjugates that address multiple receptor expressions within heterogeneous tumors .

What are the critical factors affecting FITC conjugation to c-Met antibodies, and how can researchers optimize conjugation protocols?

Optimizing FITC conjugation to c-Met antibodies requires careful control of multiple parameters:

• Critical Parameters Influencing Conjugation Efficiency:

  • pH: Optimal conjugation occurs at pH 9.5, as the reaction requires deprotonated lysine residues

  • Temperature: Room temperature (20-25°C) provides optimal reaction kinetics

  • Protein concentration: 25 mg/ml initial concentration maximizes conjugation efficiency

  • Reaction time: 30-60 minutes provides optimal labeling without over-conjugation

  • FITC quality: Use high-purity FITC with minimal hydrolyzed or oxidized components

  • Buffer composition: Avoid primary amines (Tris, glycine) that compete with antibody lysines

• Fluorescein/Protein (F/P) Ratio Optimization:

  • Optimal F/P ratio typically ranges from 3.0-6.0 for most applications

  • Higher ratios may increase brightness but can impair antibody binding

  • Lower ratios may preserve binding but reduce detection sensitivity

  • Methods to determine F/P ratio:

    • Spectrophotometric measurement at 280 nm (protein) and 495 nm (FITC)

    • Mass spectrometry to determine precise number of FITC molecules per antibody

• Step-by-Step Optimization Protocol:
  a) Antibody preparation:

  • Purify antibody by protein A/G chromatography

  • Buffer exchange to carbonate/bicarbonate buffer (pH 9.0-9.5)

  • Concentrate to 20-25 mg/ml

  b) Conjugation reaction:

  • Dissolve FITC in anhydrous DMSO (10 mg/ml)

  • Add FITC solution dropwise to antibody with gentle stirring

  • Incubate at room temperature for 30-60 minutes in darkness

  c) Purification:

  • Remove unconjugated FITC using gel filtration (e.g., Sephadex G-25)

  • Alternative: Use ultrafiltration with appropriate molecular weight cutoff

  • Analyze fractions spectrophotometrically to identify conjugated antibody

• Quality Control Assessments:

  • Determine protein recovery (typically >90% with optimal protocols)

  • Calculate F/P ratio using absorption measurements:
    F/P = (A495 × dilution factor) / [(A280 - 0.35 × A495) × 1.4]

  • Verify binding activity using known c-Met positive cells

  • Check stability through accelerated storage conditions

• Commercial Kit Adaptation for c-Met Antibodies:
  The Mix-n-Stain™ FITC Antibody Labeling Kit provides a convenient alternative:

  • Compatible with various antibody formulations (including those containing BSA)

  • Minimal hands-on time (<30 seconds)

  • Total reaction time of 15 minutes

  • No purification step required with 100% antibody recovery

Carefully optimized conjugation protocols ensure consistent performance in downstream applications while maintaining the critical binding properties of the original c-Met antibody.

How do researchers troubleshoot discrepancies between c-Met detection methods (flow cytometry versus Western blotting)?

Researchers frequently encounter discrepancies between c-Met detection methods, which can be systematically addressed:

• Understanding Common Discrepancies:
  Several c-Met antibodies show differential performance between applications:

  • seeMet 2 and 13 demonstrate strong native c-Met binding in flow cytometry but poor performance in Western blotting

  • seeMet 11 and 12 excel in Western blotting but show weak binding in flow cytometry

  These differences reflect fundamental distinctions in what each method detects:

  Parameter	Flow Cytometry	Western Blotting
  Protein State	Native conformation	Denatured linear epitopes
  Epitope Accessibility	Surface-exposed regions	All protein regions
  c-Met Form Detected	Primarily mature receptor	Both precursor and processed forms
  Quantification	Relative expression levels	Semi-quantitative band intensity
  Single-Cell Resolution	Yes	No (population average)

• Root Cause Analysis:

  • Epitope conformation: Some antibodies recognize conformational epitopes disrupted by denaturation

  • Protein processing: Differences in detecting precursor (170 kDa) vs. mature β-chain (145 kDa)

  • Post-translational modifications: Glycosylation or phosphorylation may affect antibody binding

  • Temperature sensitivity: Antibodies like seeMet 2 show dramatically reduced binding at 4°C

  • Fixation effects: Chemical fixatives can modify epitopes differently

• Methodological Solutions:
  a) For antibodies working well in flow cytometry but poorly in Western blotting:

  • Try native/non-denaturing gel electrophoresis

  • Use milder detergents or lower SDS concentrations

  • Optimize sample preparation to preserve critical epitopes

  • Consider dot blot as alternative to Western blot

  b) For antibodies working well in Western blotting but poorly in flow cytometry:

  • Optimize fixation and permeabilization for intracellular epitopes

  • Try different cell dissociation methods that better preserve surface epitopes

  • Adjust antibody incubation temperature (especially for temperature-sensitive antibodies)

  • Implement signal amplification strategies

• Validation and Cross-Confirmation Strategies:

  • Use multiple antibody clones recognizing different epitopes

  • Implement orthogonal detection methods (immunoprecipitation, ELISA)

  • Include appropriate positive and negative control cell lines

  • Use siRNA/shRNA knockdown to confirm specificity

  • Correlate with mRNA expression data

• Case Study from Research:
  The characterization of seeMet antibodies revealed:

  • Epitope mapping identified 10 different antibody binding regions across the c-Met α-chain

  • Most antibodies could immunoprecipitate the 170 kDa precursor c-Met and the 145 kDa mature β-chain

  • Flow cytometry performance classified antibodies into strong, intermediate, and weak binders

  • This comprehensive characterization allowed selection of optimal antibodies for specific applications

By understanding the molecular basis for these discrepancies and implementing appropriate methodological adjustments, researchers can select the optimal c-Met antibodies for their specific experimental needs and properly interpret potentially conflicting results.

How are c-Met FITC antibodies being utilized in the development of next-generation antibody-drug conjugates?

c-Met FITC antibodies are playing crucial roles in the development pipeline for antibody-drug conjugates (ADCs) targeting c-Met:

• Epitope Screening and Selection:

  • FITC-conjugated antibodies help identify optimal binding epitopes that:

    • Promote efficient internalization

    • Avoid triggering unwanted c-Met signaling activation

    • Target cancer-specific conformations of c-Met

  • Flow cytometric screening with c-Met-FITC antibodies allows rapid assessment of binding to diverse cell types and correlation with ADC efficacy

• Internalization and Trafficking Studies:

  • Researchers use c-Met-FITC antibodies to:

    • Quantify internalization rates under various conditions

    • Track intracellular trafficking pathways

    • Assess lysosomal delivery efficiency (critical for cleavable linker ADCs)

    • Monitor receptor recycling versus degradation

  • Time-course imaging with c-Met-FITC helps optimize the linker-payload chemistry based on trafficking patterns

• Novel ADC Design Approaches:

  • Biparatopic Antibodies:
    The METxMET biparatopic antibody approach employs two c-Met-binding domains with different epitopes, enhancing internalization and efficacy

  • Dual-Targeting Strategies:
    Research demonstrates that dual-targeting ADCs against c-Met and RON receptors (PCMdt-MMAE) can address tumor heterogeneity and potentially overcome resistance mechanisms

  • Non-Agonistic Selection:
    Antibodies like P3D12 that induce c-Met degradation with minimal signaling activation make ideal ADC candidates

• Patient Selection and Predictive Biomarkers:

  • c-Met-FITC antibodies enable:

    • Flow cytometric quantification of c-Met expression levels

    • Identification of patient subgroups likely to respond to c-Met ADCs

    • Monitoring of c-Met levels during treatment

  • Research correlates radiolabeled c-Met antibody uptake with c-Met expression and ADC efficacy

• Preclinical Development Data:
  Examples from recent research illustrate the promise of c-Met ADCs:

  ADC	Composition	Key Findings	Reference
  METxMET-M114	Biparatopic antibody with maytansinoid payload	- Effective against moderate to high c-Met expression - Maintains efficacy in TKI-resistant models - Well-tolerated in toxicology studies
  P3D12-vc-MMAF	c-Met antibody with MMAF via cleavable linker	- Active in c-Met+ cells regardless of amplification status - Effective in models where TKIs fail - Superior profile compared to c-Met pathway inhibitors
  PCMdt-MMAE	Dual MET/RON-targeting ADC with MMAE	- Addresses phenotypic heterogeneity in cancer - Favorable pharmacokinetics and safety profile - Effective against tumors with heterogeneous MET/RON

• Future Directions:

  • Development of ADCs with tumor microenvironment-activated linkers

  • Integration of imaging and therapeutic functions (theranostics)

  • Combination strategies with immune checkpoint inhibitors

  • Exploration of novel payloads beyond traditional cytotoxics

The use of c-Met FITC antibodies has been instrumental in characterizing antibody binding, internalization, and trafficking properties that directly inform the design of more effective c-Met-targeted ADCs for cancer therapy.