Phospho-ALK (Y1507) Antibody

What is the biological significance of ALK phosphorylation at Y1507?

Phosphorylation of ALK at Y1507 (corresponding to Y567 in NPM-ALK fusion proteins) serves as a critical docking site for recruiting adapter proteins including Shc, FRS2-α, and FRS2-β. This phosphorylation event initiates signal transduction from activated ALK to multiple downstream pathways including RAS/MAPK, JAK/STAT, PI3K/Akt, and PLC-γ pathways . Functionally, Y1507 phosphorylation plays a crucial role in promoting cell growth, survival, and differentiation in both physiological neural development and pathological conditions, particularly in ALK-driven cancers . Research has demonstrated that when ALK is activated either through ligand binding or oncogenic fusion events, Y1507 phosphorylation facilitates the assembly of signaling complexes that regulate multiple cellular processes.

How does Y1507 phosphorylation coordinate with other ALK phosphorylation sites?

ALK contains multiple phosphorylation sites that orchestrate signaling specificity and amplitude. While Y1507 primarily mediates interactions with Shc and FRS adaptors leading to MAPK pathway activation, it functions in concert with other critical sites. The activation loop phosphorylation sites (Y1278, Y1282, and Y1283) initiate kinase activity . Phosphoproteomic analyses reveal that in ALK-amplified neuroblastoma, Y1507 phosphorylation correlates strongly with JNK pathway activation . Research using doxycycline-inducible ALK expression systems has measured up to 38.9-fold increases in Y1507 phosphorylation following induction, confirming the importance of this site in ALK activation . Understanding this coordinated phosphorylation profile is essential when designing experiments targeting specific ALK-dependent pathways.

What are the optimal conditions for detecting Phospho-ALK (Y1507) in Western blotting?

For optimal Western blot detection of Phospho-ALK (Y1507), researchers should use a 1:1000 antibody dilution as recommended by technical datasheets . Cell lysis procedures must incorporate phosphatase inhibitors to preserve phosphorylation status. For gel separation, 6-8% SDS-PAGE gels are optimal for resolving full-length ALK (220 kDa), while 10% gels work better for ALK fusion proteins (80-90 kDa) . Blocking with 5% BSA rather than milk is essential, as milk contains phosphatases that may reduce signal. Validation should include appropriate positive controls (ALK-amplified neuroblastoma cell lines or ALK fusion-positive cell lines) and negative controls (ALK inhibitor-treated cells) . This methodology enables detection of both full-length ALK (220 kDa) and fusion proteins like NPM-ALK (80 kDa).

How should researchers apply Phospho-ALK (Y1507) antibodies in cell-based assays?

For cell-based ELISA applications, cells should be cultured directly in 96-well plates to achieve consistent density. According to protocol recommendations, cells should be fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 . A concentration of 1:100 for primary Phospho-ALK (Y1507) antibody is typically recommended, followed by HRP-conjugated secondary antibody detection . Normalization to total cell number using crystal violet staining is crucial to adjust for plating differences. For immunofluorescence, a 1:400 dilution is optimal after appropriate fixation and permeabilization . Both techniques benefit from parallel staining with total ALK antibodies to calculate phospho/total ratios for more accurate quantification of activation status.

What controls are essential when working with Phospho-ALK (Y1507) antibodies?

Comprehensive controls are critical for accurate interpretation of Phospho-ALK (Y1507) experiments. Essential positive controls include:

Researchers should also validate antibody specificity through peptide competition assays or phospho-specific immunodepletion. For quantitative studies, recombinant phosphorylated ALK proteins can serve as standards for calibration curves .

How do researchers distinguish between full-length ALK and fusion proteins using Phospho-ALK (Y1507) antibodies?

Distinguishing between phosphorylated Y1507 in full-length ALK (220 kDa) versus fusion proteins like NPM-ALK (80 kDa) requires careful experimental design. Phospho-specific antibodies targeting Y1507 recognize this site in both the full-length receptor and fusion proteins because the kinase domain containing this residue is preserved in oncogenic ALK fusions . When performing Western blot analysis, researchers should observe distinct molecular weight bands: approximately 220 kDa for full-length ALK and lower molecular weights for fusion proteins . Cell line selection is crucial - using controls like H3122 cells (EML4-ALK positive) or SU-DHL-1 cells (NPM-ALK positive) alongside ALK-negative cell lines helps validate specificity . For ambiguous results, immunoprecipitation with ALK fusion-specific antibodies followed by phospho-Y1507 detection can provide definitive identification.

How can Phospho-ALK (Y1507) antibodies contribute to phosphoproteomic studies?

Integrating Phospho-ALK (Y1507) antibodies into phosphoproteomic workflows provides powerful insights into ALK-dependent signaling networks. One sophisticated approach involves utilizing these antibodies to confirm ALK activation status before or in parallel with mass spectrometry-based phosphoproteomic profiling . Studies employing this approach have quantified over 20,000 phosphorylation sites alongside specific ALK phosphorylation events, allowing computational inference of kinase activity networks . Advanced applications include using Phospho-ALK (Y1507) antibodies for phosphotyrosine immunoprecipitation followed by mass spectrometry to enrich for ALK-specific signaling complexes . Research has demonstrated that this approach can identify direct ALK substrates when combined with in vitro kinase assays. Comparing phosphoproteomic data from samples with confirmed Y1507 phosphorylation against those treated with ALK inhibitors enables the construction of ALK-specific kinase-substrate relationship networks .

What role does monitoring Phospho-ALK (Y1507) play in studying ALK inhibitor resistance?

Monitoring Phospho-ALK (Y1507) provides critical insights into resistance mechanisms against ALK inhibitors in cancer models. Studies have shown that persistent Y1507 phosphorylation despite inhibitor treatment can indicate incomplete target engagement or the emergence of resistance mutations in the ALK kinase domain . In ALK-positive cancers, analyzing the phosphorylation status of Y1507 before and after treatment with inhibitors like crizotinib helps distinguish between ALK-dependent and ALK-independent resistance mechanisms . When resistance occurs through ALK mutations, Y1507 phosphorylation typically persists despite treatment. Conversely, when resistance emerges through bypass pathways, Y1507 phosphorylation may remain suppressed while downstream signaling recovers through alternative mechanisms. Researchers use this information to rationally design combination therapies targeting both ALK and potential bypass pathways.

How do different ALK variants affect Y1507 phosphorylation patterns?

Different ALK variants exhibit distinct Y1507 phosphorylation patterns that impact downstream signaling. Research has demonstrated that certain mutations like ALK LF655del exhibit decreased Y1507 phosphorylation compared to wild-type ALK when overexpressed, suggesting altered regulation mechanisms . Similarly, fusions like EML4-ALK show differential phosphorylation kinetics at Y1507 compared to full-length ALK . These differences in phosphorylation patterns correlate with varying responses to ALK ligands (ALKAL1/2) and inhibitors, with some variants showing resistance patterns . For robust comparisons of phosphorylation across variants, researchers should normalize phospho-Y1507 signal to total ALK expression levels and conduct parallel assessment of multiple phosphorylation sites. This approach has revealed that ALK fusion proteins in synovial sarcoma exhibit exceptionally high Y1507 phosphorylation levels compared to wild-type ALK, correlating with enhanced oncogenic activity .

How can multiplexed detection of ALK phosphorylation sites improve research outcomes?

Multiplexed detection of ALK phosphorylation sites provides a more comprehensive understanding of ALK activation status than single-site analysis. Recent research demonstrates that simultaneous assessment of Y1507, Y1278, and Y1604 phosphorylation creates signature patterns that correlate with specific ALK variants and activation states . This approach is particularly valuable in:

Advanced techniques like mass spectrometry-based multiphosphorylation site profiling have revealed that different ALK alterations (amplification, mutation, or fusion) display characteristic phosphorylation signatures across multiple sites . This multiplexed approach transforms phosphorylation analysis from a binary readout to a nuanced profile that better reflects ALK biology.

What are common sources of false positives/negatives when using Phospho-ALK (Y1507) antibodies?

Researchers must be vigilant about potential sources of false results when using Phospho-ALK (Y1507) antibodies. Common sources of false positives include cross-reactivity with other phosphorylated tyrosine kinases, particularly those with similar sequence motifs around the phosphorylation site . Inadequate blocking or insufficient washing can also generate non-specific signals. False negatives frequently result from sample handling issues - phosphorylation is labile and can be rapidly lost if phosphatase inhibitors are omitted or if samples experience extended processing times . Another major source of false negatives is epitope masking, where protein interactions or conformational changes block antibody access to the phosphorylated Y1507 site. To mitigate these issues, researchers should perform phosphatase treatment controls, use confirmed positive and negative cell lines for validation, and optimize sample preparation protocols to preserve phosphorylation status.

How should researchers interpret variability in Phospho-ALK (Y1507) detection across samples?

Interpreting variability in Phospho-ALK (Y1507) detection across samples requires considering both biological and technical factors. Biologically, samples with different ALK alterations (amplification, mutation, or fusion) exhibit varying baseline levels of Y1507 phosphorylation . Studies have shown that ALK-amplified neuroblastoma samples typically display higher Y1507 phosphorylation than samples with point mutations. From a technical perspective, pre-analytical variables significantly impact phosphorylation detection - samples must be processed rapidly with immediate fixation or snap-freezing . For quantitative comparisons, researchers should normalize phospho-signals to total ALK protein levels and employ appropriate controls. When analyzing phosphorylation across multiple experiments, researchers should consider using reference standards and calculating relative phosphorylation ratios rather than relying on absolute signal intensity, which can vary between experimental batches.