Phospho-ABL1 (Tyr204) Antibody

What is the significance of ABL1 phosphorylation at Tyr204?

ABL1 (Abelson murine leukemia viral oncogene homolog 1) phosphorylation at Tyr204 represents a critical regulatory event in ABL1 kinase activation. This specific phosphorylation site lies within the activation loop region (amino acids 156-205) and contributes to the conformational changes necessary for full kinase activity . In normal cells, ABL1 Tyr204 phosphorylation participates in regulating cell adhesion, motility, and cytoskeletal remodeling. In pathological conditions, particularly in chronic myeloid leukemia (CML), phosphorylation at this site contributes to the constitutive activation of the BCR-ABL1 fusion protein that drives leukemic cell proliferation and survival .

How does Phospho-ABL1 (Tyr204) differ from other ABL1 phosphorylation sites?

While ABL1 contains multiple phosphorylation sites, including Tyr245, Tyr412, and Thr735, each site has distinct functional implications:

Research indicates that while Tyr412 is often considered the primary marker of full ABL1 activation, Tyr204 phosphorylation occurs earlier in the activation sequence and may serve as a more sensitive indicator of initial kinase activation events .

What are the recommended applications for Phospho-ABL1 (Tyr204) antibodies?

Phospho-ABL1 (Tyr204) antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Recommended dilutions range from 1:500-1:2000

• Immunohistochemistry (IHC): Optimal at 1:100-1:300 dilutions

• ELISA: Highest sensitivity at 1:5000 dilution

• Immunofluorescence (IF): Effective at 1:50-1:200 dilutions

For optimal results in IHC applications, high-pressure and temperature Tris-EDTA (pH 8.0) antigen retrieval is recommended based on validation studies with human brain tissue samples .

How should I design validation experiments for Phospho-ABL1 (Tyr204) antibodies?

A comprehensive validation strategy should include:

• Phospho-peptide competition assay: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides. This should abolish signal with the phosphopeptide but not with the non-phosphopeptide .

• Phosphatase treatment control: Treat half of your protein sample with lambda phosphatase to remove phosphorylation, which should eliminate signal from phospho-specific antibodies.

• Kinase inhibitor treatment: In cells expressing BCR-ABL1, treatment with TKIs like imatinib or dasatinib should reduce Tyr204 phosphorylation. For example, Western blot analysis of lysates from COS7 cells treated with Adriamycin (0.5μg/ml for 24h) shows reduced phosphorylation after TKI treatment .

• Selective knockdown/knockout: Using siRNA against ABL1 should reduce total signal if the antibody is specific.

• Phospho-ELISA: Compare reactivity with phosphorylated versus non-phosphorylated immunogen peptides to confirm phospho-specificity .

What controls should be included when using Phospho-ABL1 (Tyr204) antibodies?

For rigorous experimental design, include these controls:

• Positive control: Lysate from cells known to express phosphorylated ABL1 (e.g., K562 cells for BCR-ABL1 or COS7 cells treated with Adriamycin)

• Negative control:

  • Antibody pre-absorbed with immunogen peptide

  • Cells treated with ABL1 inhibitors like imatinib

  • Non-expressing cell lines (validate with total ABL1 antibody)

• Loading control: GAPDH or β-actin for protein normalization

• Phosphorylation normalization: Total ABL1 antibody to determine phosphorylation/total protein ratio

How can I optimize signal detection for low abundance Phospho-ABL1 (Tyr204)?

When detecting low levels of phosphorylated ABL1:

• Sample preparation optimization:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Process samples rapidly at 4°C

  • Use freshly prepared lysates when possible

• Signal enhancement strategies:

  • Enrich phosphoproteins using TiO₂ enrichment as demonstrated in phosphoproteomic studies (90% for phosphopeptide enrichment, 10% for total proteome)

  • Increase antibody incubation time (overnight at 4°C)

  • Use high-sensitivity detection systems (e.g., chemiluminescent substrates with extended duration)

  • Consider using cell-based ELISA formats for quantitative detection of phosphorylation changes

• Reduce background interference:

  • Optimize blocking conditions (5% BSA in TBST is often more effective than milk for phospho-antibodies)

  • Include additional washing steps with higher stringency buffers

How can Phospho-ABL1 (Tyr204) antibodies be used to study BCR-ABL1 signaling networks?

Phospho-ABL1 (Tyr204) antibodies enable sophisticated analyses of BCR-ABL1 signaling:

• Protein complex identification: Recent research has identified a previously undisclosed MEK1/2/BCR::ABL1/BCR/ABL1 pentameric complex where MEK1/2 induces phosphorylation of multiple ABL1 residues, including Y204, affecting BCR's tumor suppressor functions and ABL1 localization .

• Pathway crosstalk analysis: Studying relationships between ABL1 phosphorylation and other signaling pathways (RAS/MAPK, PI3K/AKT) can be performed using co-immunoprecipitation followed by phospho-specific detection .

• Proximity-based signaling studies: Techniques like ABA-induced proximity system have been employed to study BCR-ABL1 interactions with phosphatases like SHP1, revealing mechanistic insights into tyrosine phosphorylation regulation .

• Temporal phosphorylation dynamics: Time-course experiments with Phospho-ABL1 (Tyr204) antibodies can reveal activation sequence following stimulation, as demonstrated in neurotrophic receptor tyrosine kinase signaling studies .

What is the relationship between MEK1/2 signaling and ABL1 Tyr204 phosphorylation?

Recent research has revealed a complex relationship:

• MEK1/2 can form a pentameric complex with BCR::ABL1, BCR and ABL1 proteins .

• This complex formation leads to phosphorylation of BCR and BCR::ABL1 at Tyr360 and Tyr177, and ABL1 at Thr735 and Tyr412, affecting:

  • Loss of BCR's tumor-suppression functions

  • Enhanced oncogenic activity of BCR::ABL1

  • Cytoplasmic retention of ABL1

  • Drug resistance in leukemic cells

• Pharmacological MEK1/2 inhibition (e.g., with Mirdametinib) causes:

  • Dissociation of the pentameric complex

  • Dephosphorylation of BCR Y360/Y177 and ABL1 Y412/T735

  • Rescue of BCR's anti-oncogenic activities

  • Nuclear accumulation of ABL1 with tumor-suppressive functions

  • Growth inhibition of leukemic cells

  • Sensitization to arsenic trioxide (ATO) therapy

This MEK1/2-ABL1 signaling axis represents a potential therapeutic target for TKI-resistant leukemia, highlighting the importance of studying ABL1 phosphorylation dynamics.

How can Phospho-ABL1 (Tyr204) antibodies contribute to resistance mechanism studies in leukemia?

These antibodies provide valuable tools for investigating TKI resistance:

• Mapping alternative activation mechanisms: In TKI-resistant cells, phosphorylation patterns at Tyr204 can reveal bypass mechanisms that maintain ABL1 activation despite inhibitor binding to the ATP pocket .

• Compound screening: Cell-based ELISA assays using Phospho-ABL1 (Tyr204) antibodies allow high-throughput screening of compounds that may overcome resistance by targeting phosphorylation at this site .

• Biomarker identification: Phospho-ABL1 (Tyr204) levels can potentially serve as biomarkers for early detection of emerging resistance before clinical manifestation .

• Combination therapy evaluation: Studies combining MEK1/2 inhibitors with ATO demonstrated enhanced survival in mice with BCR::ABL1-T315I-induced leukemia, with Phospho-ABL1 antibodies helping to elucidate the mechanism .

Why might I observe multiple bands in Western blots using Phospho-ABL1 (Tyr204) antibody?

Multiple bands can occur for several reasons:

• Multiple ABL1 isoforms: ABL1 exists in multiple isoforms (1a and 1b) with different molecular weights.

• BCR-ABL1 fusion proteins: In cells expressing BCR-ABL1 fusions (common in CML), you may observe bands at ~210 kDa (p210 BCR-ABL1) or ~190 kDa (p190 BCR-ABL1) in addition to native ABL1 at ~125-145 kDa .

• Proteolytic degradation: Incomplete protease inhibition during sample preparation can result in fragmentation.

• Cross-reactivity: Some antibodies may cross-react with other phosphorylated tyrosine kinases, though high-quality Phospho-ABL1 (Tyr204) antibodies show minimal cross-reactivity with other proteins .

• Post-translational modifications: Ubiquitination or SUMOylation can cause higher molecular weight bands.

Validation with blocking peptides can help determine which bands represent specific recognition of phosphorylated ABL1 .

How can I distinguish between phosphorylation of BCR-ABL1 and c-ABL1 in leukemic cells?

Distinguishing between these phosphorylated proteins requires careful experimental design:

• Molecular weight discrimination: Native ABL1 (~125-145 kDa) versus BCR-ABL1 fusion proteins (~190-210 kDa) can be distinguished by size on Western blots .

• Sequential immunoprecipitation:

  • First IP with BCR antibody, then blot with Phospho-ABL1 (Tyr204) for BCR-ABL1

  • IP the supernatant with ABL1 antibody, then blot with Phospho-ABL1 (Tyr204) for native ABL1

• Subcellular fractionation: BCR-ABL1 is predominantly cytoplasmic, while c-ABL1 shuttles between cytoplasm and nucleus .

• Use of knockout/knockdown models: Selective knockdown of BCR-ABL1 using targeted siRNAs can help differentiate signals.

• Peptide biosensor assays: Novel ELISA-based peptide biosensor assays have been developed that can detect ABL1 kinase activity with high specificity, distinguishing between different forms of ABL1 .

What factors affect the phosphorylation state of ABL1 at Tyr204 during sample preparation?

Several factors can artificially alter ABL1 phosphorylation status:

• Time and temperature: Delayed processing or higher temperatures activate endogenous phosphatases.

• Phosphatase inhibitor cocktail composition: Must include:

  • Tyrosine phosphatase inhibitors (sodium orthovanadate)

  • Serine/threonine phosphatase inhibitors (sodium fluoride, β-glycerophosphate)

  • Broad-spectrum phosphatase inhibitors (sodium pyrophosphate)

• Cell lysis method: Harsh detergents may disrupt protein-protein interactions that protect phosphorylation sites.

• Cell culture conditions: Serum starvation, cell density, and stress can all affect basal phosphorylation.

• Drug treatments: Exposure to tyrosine kinase inhibitors like imatinib rapidly reduces phosphorylation at Tyr204 .

For optimal phospho-preservation, lyse cells directly in hot SDS-PAGE sample buffer containing phosphatase inhibitors or use specialized phosphoprotein preservation buffers.

How can Phospho-ABL1 (Tyr204) antibodies contribute to personalized medicine approaches for leukemia?

These antibodies enable several personalized medicine strategies:

• Prediction of TKI response: Baseline and early-treatment phosphorylation levels at Tyr204 may predict response to TKIs in individual patients .

• Resistance mechanism identification: Patients with persistent Tyr204 phosphorylation despite TKI treatment may harbor specific resistance mechanisms that could guide subsequent therapy selection .

• Novel combination strategies: The MEK1/2-ABL1 signaling axis discovery suggests combining MEK inhibitors with standard therapies for resistant disease .

• Patient-derived cell models: Phospho-ABL1 (Tyr204) antibodies can be used to characterize phosphorylation patterns in patient-derived xenografts or primary cell cultures to develop personalized treatment approaches .

What novel methodologies are being developed for monitoring ABL1 Tyr204 phosphorylation?

Several innovative approaches are emerging:

• ELISA-based peptide biosensors: These assays utilize ABL1-specific peptide sequences to detect kinase activity with high specificity, providing an alternative to traditional antibody-based methods .

• Proximity-dependent labeling techniques: Methods that enable detection of protein-protein interactions in the context of ABL1 signaling complexes .

• Mass spectrometry-based phosphoproteomics: Large-scale identification of phosphorylation changes across the proteome, including at ABL1 Tyr204, provides comprehensive signaling pathway analysis .

• Chemical-induced proximity systems: Novel approaches using ABA (abscisic acid)-induced proximity to study the function of BCR::ABL1 and its interaction with phosphatases .

• Temporal phosphoproteomics: Time-resolved studies of phosphorylation dynamics following receptor activation, enabling more comprehensive understanding of signaling network behavior .

How do phosphorylation patterns at Tyr204 and other sites integrate into ABL1 signaling networks?

Research reveals complex interrelationships:

• Hierarchical phosphorylation: Evidence suggests a specific sequence of phosphorylation events, with Tyr204 potentially serving as an early indicator of ABL1 activation before Tyr412 phosphorylation .

• Cross-regulation with other modifications: Phosphorylation at Tyr204 may influence other post-translational modifications (ubiquitination, SUMOylation) that regulate ABL1 stability and localization .

• Allosteric effects on protein interactions: Phosphorylation status affects ABL1's ability to form complexes with other signaling proteins (MEK1/2, BCR) and influences subcellular localization .

• Feedback regulation: Phosphorylated ABL1 can activate phosphatases that ultimately regulate its own activity, creating complex feedback loops .

• Integration with multiple pathways: Phosphorylated ABL1 participates in diverse cellular processes including cytoskeletal regulation, DNA damage response, and apoptosis, with phosphorylation patterns dictating pathway specificity .