Phospho-MET (Tyr1234) Antibody

What is Phospho-MET (Tyr1234) Antibody and what does it specifically detect?

Phospho-MET (Tyr1234) Antibody is a specialized immunological tool designed to recognize c-MET receptor tyrosine kinase only when phosphorylated at tyrosine residue 1234. This phosphorylation site, often in conjunction with Tyr1235, is located within the activation loop of the kinase domain and represents a critical regulatory event in c-MET signaling. When hepatocyte growth factor (HGF) binds to c-MET, it triggers receptor dimerization and autophosphorylation at these sites, initiating downstream signaling cascades. The antibody's specificity allows researchers to monitor the activation state of c-MET in experimental systems, making it valuable for studying both normal physiological processes and pathological conditions where aberrant c-MET signaling occurs .

Some commercially available antibodies detect only phosphorylation at Tyr1234, while others recognize dual phosphorylation at both Tyr1234 and Tyr1235. This distinction is important when designing experiments, as single-site phosphorylation may have different biological implications than dual-site phosphorylation .

What are the typical applications for Phospho-MET (Tyr1234) Antibody in research?

Phospho-MET (Tyr1234) antibodies have been validated for multiple research applications, making them versatile tools for investigating c-MET activation across different experimental systems:

These applications allow researchers to investigate c-MET activation in various contexts, from cell culture systems to animal models and human tissue samples. The choice of application depends on the specific research question, with Western blotting being the most widely validated technique for phospho-MET detection .

What is the biological significance of c-MET phosphorylation at Tyr1234?

c-MET phosphorylation at Tyr1234 represents a critical regulatory event in receptor activation and downstream signaling. The phosphorylation of this residue in the activation loop of the kinase domain enables catalytic activity of the receptor. After HGF binding and receptor dimerization, c-MET undergoes autophosphorylation at multiple tyrosine residues, with Tyr1234/1235 phosphorylation being essential for kinase activity .

This phosphorylation event triggers interaction with multiple downstream signaling molecules including PI3-kinase subunit PIK3R1, PLCG1, SRC, GRB2, and STAT3 . These interactions activate several signaling cascades:

• RAS-ERK pathway - promoting cell proliferation and gene expression

• PI3-kinase-AKT pathway - supporting cell survival and metabolism

• PLCγ-PKC pathway - regulating cellular migration and morphogenesis

The activated c-MET receptor plays crucial roles in both embryonic development (gastrulation, muscle and neuron development, angiogenesis, kidney formation) and adult physiological processes (wound healing, organ regeneration, tissue remodeling) . Aberrant activation through mutation, overexpression, or inappropriate ligand stimulation is linked to various cancers including gastric, renal, and breast malignancies .

How should I prepare cell samples to effectively detect Phospho-MET (Tyr1234)?

Proper sample preparation is critical for preserving phosphorylation states, which can be rapidly lost due to phosphatase activity. For optimal phospho-MET detection, follow these methodological guidelines:

• Cell stimulation protocol:

  • Culture cells to 70-80% confluence to ensure optimal receptor expression

  • Serum-starve cells for 24-48 hours to reduce baseline phosphorylation

  • Stimulate with recombinant HGF (typically 10-50 ng/mL for 10 minutes) to induce Met phosphorylation

• Lysis procedure:

  • Rapidly lyse cells in ice-cold lysis buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Include protease inhibitors to prevent protein degradation

  • Keep samples cold throughout processing to minimize phosphatase activity

  • Clarify lysates by centrifugation (14,000 × g, 15 minutes, 4°C)

• For immunoprecipitation:

  • Use 1:50 dilution of phospho-Met antibody or total Met antibody (e.g., DO-24)

  • Process immunoprecipitated samples immediately for Western blotting or other analyses

  • Follow with anti-phosphotyrosine detection or specific phospho-Met antibody

This methodological approach ensures preservation of the phosphorylation state, enabling reliable detection of activated c-MET in experimental systems.

What controls should I include when using Phospho-MET (Tyr1234) Antibody?

Including appropriate controls is essential for validating results obtained with phospho-specific antibodies. For Phospho-MET (Tyr1234) experiments, incorporate these critical controls:

• Positive controls:

  • HGF-stimulated cells known to express c-MET (e.g., A549 human lung carcinoma cells treated with 10-50 ng/mL HGF for 10 minutes)

  • Cell lines with constitutively active c-MET signaling (certain cancer cell lines)

• Negative controls:

  • Unstimulated/serum-starved cells to establish baseline phosphorylation

  • Cells treated with c-MET inhibitors to demonstrate signal specificity

  • Phosphatase-treated lysates to confirm phosphorylation dependency of signal

• Antibody specificity controls:

  • Peptide competition assays using specific phosphopeptides corresponding to phospho-Tyr1234, phospho-Tyr1235, or dual phospho-Tyr1234/1235

  • Comparison between antibodies targeting different phosphorylation sites

• Loading and normalization controls:

  • Total c-MET antibody (e.g., DL-21) to normalize for receptor expression levels

  • Housekeeping proteins (β-actin, GAPDH) for total protein loading

For Western blot applications, consider running treated and untreated samples side by side, allowing direct comparison of phosphorylation states on the same membrane . For immunohistochemistry, include both positive and negative tissue controls to validate staining patterns .

How do I optimize antibody dilution and incubation conditions for different applications?

Optimizing antibody conditions is essential for obtaining specific signals with minimal background. Follow these application-specific guidelines:

For Western Blotting:

• Start with the manufacturer's recommended dilution, typically 1:1000

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000) to determine optimal concentration

• Optimize incubation time and temperature (typically overnight at 4°C or 2 hours at room temperature)

• Use 5% BSA in TBST as blocking agent rather than milk (which contains phosphatases)

• Include phosphatase inhibitors in all buffers when possible

For Immunoprecipitation:

• Use a more concentrated antibody dilution, typically 1:50

• Optimize protein A/G bead amount and incubation time

• Consider pre-clearing lysates to reduce non-specific binding

• Wash stringently to remove non-specific interactions

For Immunohistochemistry/Immunofluorescence:

• Optimize antigen retrieval methods (heat-induced epitope retrieval often works best)

• Test different fixation protocols as phospho-epitopes can be sensitive to fixation

• Determine optimal antibody concentration through titration

• Include blocking of endogenous peroxidase activity for IHC applications

• Consider signal amplification systems for low-abundance targets

The optimal conditions will vary based on the specific antibody clone, sample type, and expression level of phospho-MET in your experimental system. Document all optimization steps for reproducibility and include optimization data in supplementary materials when publishing.

How do I properly quantify and normalize phospho-MET (Tyr1234) levels in Western blot analysis?

Rigorous quantification of phospho-MET requires careful normalization and statistical analysis:

• Image acquisition:

  • Capture images within the linear dynamic range of your detection system

  • Avoid saturated pixels which prevent accurate quantification

  • Use consistent exposure settings across experimental replicates

• Normalization strategy:

  • Primary normalization: Normalize phospho-MET signal to total MET protein level (not to housekeeping proteins)

  • Secondary normalization: Verify equal loading using housekeeping proteins (β-actin, GAPDH)

  • Express results as phospho-MET/total MET ratio to account for variations in total receptor expression

• Quantification protocol:

  • Use appropriate image analysis software (ImageJ, Image Lab, etc.)

  • Subtract local background for each band

  • Define regions of interest consistently across all samples

  • Measure integrated density rather than peak intensity

• Statistical analysis:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Report phosphorylation as fold-change relative to control conditions

  • Include error bars representing standard deviation or standard error

For comparison across multiple blots, include a common reference sample on each blot as an internal standard. This approach enables more accurate comparison of samples analyzed on different blots or on different days.

How do I distinguish between specific phospho-MET signal and non-specific artifacts?

Differentiating genuine phospho-MET signal from technical artifacts requires careful experimental design and validation:

• Verify correct molecular weight:

  • Phospho-MET should appear at approximately 145 kDa in Western blots

  • Be aware that partially processed forms or proteolytic fragments may appear at different molecular weights

  • Some phospho-MET antibodies may cross-react with phosphorylated Src proteins in Western blot

• Signal validation approaches:

  • Compare the signal pattern with that of total MET antibody

  • Perform kinetics experiments (HGF stimulation time course)

  • Test signal reduction upon phosphatase treatment

  • Verify signal reduction after c-MET inhibitor treatment

  • Confirm signal reduction after c-MET knockdown/knockout

• Address potential artifacts:

  • Non-specific bands: Document their molecular weight and consistency

  • High background: Optimize blocking conditions and washing steps

  • Variable signal: Standardize lysate preparation and handling

  • Signal in presumed negative controls: Consider basal activation or antibody cross-reactivity

• Cross-validation with alternative techniques:

  • Verify key findings using a different phospho-MET antibody

  • Confirm activation using downstream signaling markers (phospho-ERK, phospho-AKT)

  • Consider orthogonal techniques (mass spectrometry) for critical findings

Remember that some antibodies specifically designed for phospho-Tyr1234 may show different reactivity patterns compared to dual phospho-Tyr1234/1235 antibodies . Understanding these differences is crucial for accurate interpretation of experimental results.

How do phospho-MET (Tyr1234) levels correlate with c-MET receptor activation and biological outcomes?

Understanding the relationship between phospho-MET levels and biological responses requires consideration of several factors:

• Activation threshold effects:

  • Low-level phosphorylation may be insufficient to trigger full biological responses

  • Different downstream pathways may have different activation thresholds

  • Quantitative correlation between phosphorylation level and biological effect is often non-linear

• Temporal dynamics:

  • Peak phosphorylation typically occurs within 10-30 minutes of HGF stimulation

  • Biological responses (proliferation, migration) manifest over longer timeframes

  • Sustained low-level phosphorylation may have different effects than transient high-level phosphorylation

• Spatial considerations:

  • Phosphorylated receptors in different subcellular compartments may trigger distinct signaling outcomes

  • Receptor internalization affects signaling duration and pathway specificity

  • Plasma membrane vs. endosomal signaling may activate different downstream pathways

• Experimental validation approaches:

  • Correlate phospho-MET levels with activation of immediate downstream effectors (Gab1, Grb2)

  • Measure activation of pathway-specific nodes (ERK for proliferation, AKT for survival)

  • Assess biological outcomes using functional assays (proliferation, migration, survival)

  • Use pathway inhibitors to establish causality between phosphorylation and biological effects

How can I distinguish between phosphorylation at Tyr1234 versus dual phosphorylation at Tyr1234/1235?

Distinguishing between single and dual phosphorylation states requires specific methodological approaches:

• Antibody selection:

  • Use site-specific antibodies validated for exclusive recognition of phospho-Tyr1234 or dual phospho-Tyr1234/1235

  • Some antibodies specifically recognize only the single phosphorylation state at Tyr1234

  • Others specifically detect the dual phosphorylation at both Tyr1234/1235

• Validation by ELISA specificity testing:

  • Perform comparative binding assays using synthetic peptides containing:

    • Phospho-Tyr1234 only

    • Phospho-Tyr1235 only

    • Dual phospho-Tyr1234/1235

    • Unphosphorylated control peptide

  • This approach can quantitatively demonstrate antibody specificity profiles

• Experimental verification:

  • Compare reactivity patterns using different antibodies on identical samples

  • Perform time-course experiments to detect potential sequential phosphorylation

  • Use phosphatase treatments with different specificities

  • Consider kinase assays with site-directed mutants (Y1234F or Y1235F)

An example specificity test demonstrated that some monoclonal antibodies (like HL2907) show high specificity for phospho-Tyr1234 with minimal cross-reactivity to phospho-Tyr1235 or dual phosphorylation sites . Understanding these distinctions is crucial for accurately interpreting experimental results, particularly when studying the molecular mechanisms of c-MET activation.

How can I use phospho-MET (Tyr1234) antibodies to evaluate the efficacy of MET-targeted therapies?

Phospho-MET antibodies are valuable tools for assessing drug efficacy in preclinical and clinical studies:

• In vitro drug evaluation:

  • Measure dose-dependent inhibition of MET phosphorylation using Western blotting

  • Determine IC50 values for MET phosphorylation vs. biological outcomes

  • Assess durability of inhibition through time-course experiments

  • Correlate phosphorylation inhibition with downstream pathway suppression

• In vivo efficacy assessment:

  • Analyze tumor sections using immunohistochemistry with anti-phospho-MET antibodies

  • Correlate phospho-MET reduction with tumor growth inhibition

  • Develop pharmacodynamic/pharmacokinetic relationships

  • Associate phospho-MET levels with response durability

• Resistance mechanism investigation:

  • Monitor phospho-MET status during development of resistance

  • Determine if resistance involves restoration of phospho-MET despite drug presence

  • Identify bypass pathways activated when phospho-MET remains suppressed

  • Use in combination with genetic analysis to identify resistance mutations

• Translational applications:

  • Use as pharmacodynamic biomarkers in clinical trials

  • Evaluate on-target activity of experimental compounds

  • Assess pathway reactivation in progressing lesions

  • Guide combination therapy selection based on pathway analysis

A practical example is the analysis of tumor specimens from xenograft models, where anti-phospho-MET antibodies (Tyr 1234/1235) were used to demonstrate effective target inhibition by MET-directed therapies, with reduced phospho-MET levels correlating with decreased tumor growth .

What are the methodological considerations for studying phospho-MET in tissue microenvironments and heterogeneous samples?

Analyzing phospho-MET in complex tissue environments presents unique challenges requiring specialized approaches:

• Tissue processing and preservation:

  • Rapid fixation is critical to preserve phosphorylation status

  • Consider PAXgene or other phospho-preserving fixatives instead of standard formalin

  • Optimize tissue thickness and fixation time to maintain epitope accessibility

  • Standardize time from tissue collection to fixation across all samples

• Antigen retrieval optimization:

  • Test multiple retrieval methods (citrate, EDTA, enzymatic) to determine optimal conditions

  • Verify that retrieval conditions maintain phospho-epitope integrity

  • Balance retrieval strength with tissue morphology preservation

  • Include phospho-controls in each staining batch

• Signal specificity in heterogeneous tissues:

  • Include adjacent normal tissue as internal control when possible

  • Perform dual staining with total MET to confirm specificity

  • Consider dual staining with cell type-specific markers to identify expressing cells

  • Use phosphatase-treated serial sections as negative controls

• Quantification approaches:

  • Develop consistent scoring systems (H-score, Allred, etc.)

  • Consider digital pathology and automated image analysis

  • Account for tumor heterogeneity through multiple region sampling

  • Report both intensity and percentage of positive cells

• Special considerations for heterogeneous samples:

  • For xenografts, use species-specific antibodies to distinguish host vs. tumor signals

  • In mixed cultures, combine with lineage markers for cell-specific analysis

  • For circulating tumor cells, optimize fixation protocols for liquid samples

  • In patient-derived organoids, standardize growth conditions before phospho-analysis

These methodological refinements enable reliable assessment of phospho-MET status in complex tissue environments, crucial for translational research and biomarker development.

What should I do if I observe weak or inconsistent phospho-MET (Tyr1234) signal despite proper stimulation?

Weak or inconsistent phospho-MET signals are common challenges that can be addressed through systematic troubleshooting:

• Sample preparation issues:

  • Verify c-MET expression levels using total MET antibody

  • Ensure rapid sample processing to preserve phosphorylation

  • Increase phosphatase inhibitor concentration in all buffers

  • Optimize cell lysis conditions (detergent type/concentration)

  • Consider sample enrichment through immunoprecipitation prior to Western blot

• Stimulation protocol refinement:

  • Confirm HGF bioactivity with a known responsive cell line

  • Test different HGF concentrations (10-100 ng/mL)

  • Optimize stimulation duration (5-30 minutes)

  • Consider cell density effects on receptor activation

  • Verify serum starvation conditions to reduce baseline activation

• Antibody-related optimizations:

  • Try fresh antibody aliquots (avoid repeated freeze-thaw cycles)

  • Adjust antibody concentration based on expression level

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider alternative phospho-MET antibodies (monoclonal vs. polyclonal)

  • Verify species reactivity matches your experimental system

• Detection system enhancements:

  • Use high-sensitivity ECL substrates for Western blot

  • Increase exposure time while avoiding background development

  • Try signal amplification systems (biotin-streptavidin, tyramide)

  • Optimize secondary antibody concentration

  • Consider alternative membrane types (PVDF vs. nitrocellulose)

• Technical considerations:

  • Ensure proper transfer of high molecular weight proteins (145 kDa)

  • Use freshly prepared buffers and reagents

  • Optimize blocking conditions (BSA vs. milk proteins)

  • Consider non-reducing conditions if epitope is sensitive to reduction

Systematically addressing these factors often resolves weak signal issues and improves experimental reproducibility.

How do I address non-specific bands or high background when using phospho-MET (Tyr1234) antibodies?

Non-specific signals and high background can compromise data quality but can be mitigated through targeted approaches:

• Non-specific bands in Western blot:

  • Verify the correct molecular weight for phospho-MET (approximately 145 kDa)

  • Be aware of potential cross-reactivity with phosphorylated Src proteins

  • Increase washing stringency (more washes, higher salt concentration)

  • Try different blocking agents (5% BSA is often superior to milk for phospho-detection)

  • Consider gradient gels for better separation of high molecular weight proteins

• High background in Western blot:

  • Optimize antibody dilution (try more dilute concentrations)

  • Reduce primary antibody incubation time or temperature

  • Use highly purified antibody formats when available

  • Add 0.05-0.1% SDS to washing buffer to reduce non-specific binding

  • Check for contamination in blocking reagents or buffers

• Background in immunohistochemistry/immunofluorescence:

  • Block endogenous peroxidase activity thoroughly

  • Include species-specific serum in blocking buffer

  • Optimize antibody concentration through titration

  • Include additional blocking steps (avidin/biotin, mouse-on-mouse)

  • Consider antigen retrieval optimization

• Antibody specificity issues:

  • Validate antibody using positive and negative controls

  • Perform peptide competition assays with specific phospho-peptides

  • Consider monoclonal antibodies for higher specificity

  • Verify results with a second antibody targeting the same phospho-site

• Signal validation approaches:

  • Compare phospho-signal pattern with unstimulated controls

  • Verify signal reduction after phosphatase treatment

  • Confirm that c-MET inhibitor treatment reduces the specific band but not non-specific bands

  • Use c-MET knockdown/knockout samples as definitive controls

These strategies help distinguish specific phospho-MET signal from technical artifacts, ensuring reliable and reproducible experimental results.

What are the potential causes and solutions for contradictory results when comparing different phospho-MET antibodies?

Discrepancies between different phospho-MET antibodies can arise from several sources:

• Epitope specificity differences:

  • Some antibodies detect only phospho-Tyr1234, while others recognize dual phospho-Tyr1234/1235

  • Epitope accessibility may differ depending on protein conformation

  • Different clones may recognize slightly different amino acid sequences surrounding the phosphorylation site

  • Solution: Review the exact epitope specifications and validation data for each antibody

• Antibody format variations:

  • Monoclonal antibodies (e.g., D26, HL2907) offer higher specificity but potentially lower sensitivity

  • Polyclonal antibodies may detect multiple epitopes but with higher background

  • Solution: Match antibody format to your application needs (specificity vs. sensitivity)

• Species reactivity differences:

  • Some antibodies react with human, mouse, and rat (H M R), while others have limited species reactivity

  • Amino acid sequence variations around the phosphorylation site affect antibody binding

  • Solution: Verify species cross-reactivity claims with validation data for your species of interest

• Application-specific performance:

  • Antibodies validated for Western blot may not perform well in IHC or flow cytometry

  • Fixation sensitivity varies between antibody clones

  • Solution: Use antibodies specifically validated for your application of interest

• Technical variations in protocols:

  • Different optimal dilutions may be required (1:1000 for WB, 1:50 for IP)

  • Buffer composition affects antibody binding

  • Solution: Optimize conditions individually for each antibody rather than using identical protocols

When encountering contradictory results, consider using orthogonal approaches to validate your findings. This might include using multiple antibodies targeting different phospho-epitopes, correlating with downstream signaling events, or employing mass spectrometry for definitive phosphorylation site analysis.

How might emerging technologies enhance phospho-MET detection and analysis?

Emerging technologies are poised to revolutionize phospho-MET research:

• Advanced microscopy techniques:

  • Super-resolution microscopy for nanoscale visualization of phospho-MET clusters

  • Live-cell imaging with genetically encoded biosensors for real-time phosphorylation dynamics

  • Correlative light and electron microscopy (CLEM) for ultrastructural context of phospho-MET

  • These approaches will reveal spatial organization of phospho-MET signaling complexes at unprecedented resolution

• Mass spectrometry innovations:

  • Targeted MS approaches for absolute quantification of phosphorylation stoichiometry

  • Single-cell phosphoproteomics for heterogeneity analysis

  • Spatial mass spectrometry for in situ phosphorylation mapping in tissues

  • These methods will provide comprehensive phosphorylation profiles beyond single antibody recognition

• Multiplexed detection platforms:

  • Cyclic immunofluorescence for simultaneous detection of multiple phosphorylation sites

  • Mass cytometry (CyTOF) for high-parameter single-cell phospho-profiling

  • Digital spatial profiling for spatial context of multiple phosphorylation events

  • These technologies enable integrated pathway analysis rather than isolated phospho-site detection

• Microfluidic and organ-on-chip systems:

  • Real-time phosphorylation monitoring in physiologically relevant microenvironments

  • Gradient generation for studying spatial phospho-MET responses

  • Integration with single-cell analysis for heterogeneity assessment

  • These platforms will bridge the gap between simplified in vitro systems and complex in vivo models

• Computational approaches:

  • Machine learning for automated phospho-signal quantification in complex images

  • Integrative multi-omics to place phospho-MET in broader signaling networks

  • Predictive modeling of phosphorylation dynamics and functional outcomes

  • These computational tools will transform descriptive phospho-data into predictive models

These technological advances will enable more comprehensive, dynamic, and physiologically relevant analysis of phospho-MET signaling in health and disease.

What are the emerging research questions regarding phospho-MET (Tyr1234) in cancer and therapeutic resistance?

Critical research questions driving the field forward include:

• Heterogeneity and plasticity in phospho-MET signaling:

  • How does phospho-MET distribution vary within tumors at single-cell resolution?

  • How do microenvironmental factors dynamically regulate MET phosphorylation?

  • Can phospho-MET patterns predict inherent or acquired resistance to targeted therapies?

  • These questions address the complex reality of phospho-MET signaling in heterogeneous tumors

• Relationship between MET mutations and phosphorylation patterns:

  • How do specific mutations affect phosphorylation at Tyr1234 vs. other sites?

  • Do different mutational profiles create distinct phosphorylation signatures?

  • Can phospho-patterns predict functional outcomes of novel mutations?

  • These investigations connect genotypic variations to phospho-phenotypes

• Crosstalk with other RTK systems:

  • How does phospho-MET status influence and respond to other RTK systems (EGFR, HER2, AXL)?

  • What is the temporal sequence of phosphorylation events during resistance development?

  • Can targeting specific phosphorylation sites prevent or overcome resistance?

  • These studies address the complex signaling networks beyond isolated phospho-MET analysis

• Novel therapeutic approaches based on phosphorylation dynamics:

  • Can intermittent vs. continuous inhibition strategies prevent adaptive phospho-MET upregulation?

  • How do phosphatase modulators affect phospho-MET equilibrium compared to kinase inhibitors?

  • Can targeting downstream phosphorylation-dependent interactions overcome resistance?

  • These questions expand therapeutic strategies beyond direct kinase inhibition

• Computational modeling of phospho-MET networks:

  • Can phosphorylation patterns be used to predict therapy response through machine learning?

  • How do temporal dynamics of phosphorylation correlate with therapeutic outcomes?

  • Can integrated phospho-signatures replace single-site analysis for clinical decision-making?

  • These approaches leverage computational power to extract maximal information from phospho-data

Addressing these questions will require sophisticated experimental models and analytical approaches, including patient-derived organoids, in vivo imaging, and integrated multi-omics profiling technologies.