Phospho-ALK (Y1604) Antibody

What is the significance of ALK Y1604 phosphorylation in cellular signaling?

Phosphorylation at tyrosine 1604 of ALK (Y1604) represents a critical regulatory event in ALK signaling pathways. This specific phosphorylation site is required for the interaction between ALK and phospholipase C gamma (PLCγ), forming a crucial connection point in downstream signaling cascades. The phosphorylation at this residue is essential for the oncogenic activity of ALK fusion proteins, particularly NPM-ALK, as demonstrated through site-directed mutagenesis studies that show loss of oncogenic potential when this tyrosine is mutated . This phosphorylation event serves as a molecular switch that enables signal transduction through the MAPK pathway, leading to cellular proliferation and survival signaling, particularly in malignant conditions where ALK is dysregulated .

What are the primary applications for Phospho-ALK (Y1604) antibody in research?

Phospho-ALK (Y1604) antibodies are versatile research tools validated for multiple experimental applications. According to recent technical data, these antibodies can be effectively employed in Western blotting (WB) with recommended dilutions of 1:500-1:2000, immunoprecipitation (IP) at approximately 1:50 dilution, immunohistochemistry (IHC) at 1:100-1:300, immunofluorescence (IF) at 1:200-1:000, and ELISA at 1:5000 . These applications allow researchers to investigate ALK phosphorylation status in various experimental contexts, from protein lysates to fixed tissue samples. For protein detection, Western blotting remains the gold standard, where phospho-ALK (Y1604) appears at approximately 220 kDa for full-length ALK and 80 kDa for the NPM-ALK fusion protein . The diversity of applications makes this antibody particularly valuable for comprehensive studies involving multiple complementary techniques.

What species reactivity can researchers expect from commercially available Phospho-ALK (Y1604) antibodies?

Commercial Phospho-ALK (Y1604) antibodies demonstrate variable species reactivity profiles depending on the manufacturer and production method. Recent product specifications indicate that some antibodies, like those from Cell Signaling Technology, show specific reactivity to human ALK proteins , while other products, such as those from St John's Labs, exhibit broader cross-reactivity with human, rat, and mouse ALK proteins . This species variation is critical information for researchers working with different model systems. When selecting an antibody for cross-species experiments, researchers should carefully verify reactivity claims and potentially conduct validation experiments to confirm detection in their specific model organisms, especially when working with less common research models not explicitly listed in product specifications.

How do researchers distinguish between full-length ALK and fusion proteins when using Phospho-ALK (Y1604) antibodies?

Distinguishing between full-length ALK and various fusion proteins when using Phospho-ALK (Y1604) antibodies primarily relies on molecular weight differences in Western blot applications. Full-length ALK appears at approximately 220 kDa, while common fusion proteins present at distinctly different molecular weights – for example, NPM-ALK appears at approximately 80 kDa . In clinical research samples, EML4-ALK fusion proteins may appear at various molecular weights depending on the fusion variant. For accurate identification, researchers should include positive controls with known ALK fusion proteins alongside molecular weight markers. Additionally, supplementary techniques such as RT-PCR for fusion transcripts or using antibodies specific to fusion partners (e.g., anti-NPM or anti-EML4) in parallel experiments can provide confirmatory evidence of specific fusion protein expression rather than relying solely on phospho-specific antibody detection .

What are the optimal sample preparation conditions for preserving ALK Y1604 phosphorylation?

Preserving phosphorylation at Y1604 during sample preparation requires meticulous attention to multiple factors. First, samples should be processed rapidly post-collection, ideally flash-frozen in liquid nitrogen if immediate processing isn't possible. For cell lysis, a buffer containing strong phosphatase inhibitors is essential – typically a combination of sodium orthovanadate (1-2 mM), sodium fluoride (5-10 mM), β-glycerophosphate (10 mM), and commercial phosphatase inhibitor cocktails. The lysis buffer should maintain a slightly alkaline pH (7.5-8.0) as acidic conditions can promote dephosphorylation. For tissue samples, homogenization should be performed at 4°C using mechanical disruption methods that minimize heat generation. When working with clinical specimens, the addition of EDTA (1-2 mM) helps chelate metal ions that could activate endogenous phosphatases. Importantly, all sample processing steps from collection through analysis should maintain cold chain conditions (0-4°C) to minimize enzymatic dephosphorylation activity . For particularly challenging samples, consider specialized phospho-preservation fixatives when conducting immunohistochemistry rather than standard formalin.

How should experiments be designed to study the functional consequences of ALK Y1604 phosphorylation?

Designing experiments to study the functional consequences of ALK Y1604 phosphorylation requires a multi-faceted approach. Begin with site-directed mutagenesis to generate Y1604F (phospho-null) mutants in relevant ALK constructs (full-length or fusion proteins). These constructs, along with wild-type controls, should be expressed in appropriate cell models that lack endogenous ALK expression to avoid confounding results. Following expression, researchers should verify ALK protein levels and confirm the phosphorylation status using Phospho-ALK (Y1604) antibody by Western blotting at a dilution of 1:1000 . Downstream functional assays should include:

Additionally, transcriptome analysis comparing wild-type and Y1604F mutant cells can identify gene expression networks regulated by this phosphorylation event. For in vivo relevance, xenograft models comparing wild-type and Y1604F-expressing cells can demonstrate the importance of this phosphorylation site in tumor formation and progression .

What controls are essential when validating a new lot of Phospho-ALK (Y1604) antibody?

Thorough validation of a new Phospho-ALK (Y1604) antibody lot requires systematic implementation of positive and negative controls. Essential positive controls include cell lines with known ALK fusion proteins, such as NPM-ALK-positive anaplastic large cell lymphoma lines (e.g., SU-DHL-1, Karpas 299) or EML4-ALK-positive lung cancer lines (e.g., H3122, H2228). These should be run alongside the same cell lines treated with ALK inhibitors (e.g., crizotinib, alectinib) as negative controls to demonstrate phosphorylation-specific detection. Additionally, researchers should include:

• Peptide competition assays using the phosphorylated immunogen peptide versus the non-phosphorylated equivalent (region 1570-1619 of ALK)

• Comparison with a previously validated antibody lot on identical samples

• Phosphatase-treated lysate controls to confirm phospho-specificity

• Knockout or siRNA ALK-depleted samples as negative controls

• Overexpressed wild-type ALK versus Y1604F mutant constructs

Each new antibody lot should detect the expected molecular weights (220 kDa for full-length ALK and 80 kDa for NPM-ALK) with minimal non-specific bands. Signal intensity should correlate with known ALK expression levels across cell lines and decrease proportionally with ALK inhibitor treatment. Quantitative assessment of lot-to-lot variability should be documented to ensure experimental reproducibility over extended research timelines.

How can researchers quantitatively assess changes in ALK Y1604 phosphorylation levels?

Quantitative assessment of ALK Y1604 phosphorylation requires rigorous methodological approaches across multiple platforms. For Western blot analysis, densitometric quantification should always normalize phospho-ALK (Y1604) signal to total ALK protein rather than housekeeping proteins to account for variations in ALK expression levels between samples. For more precise quantification, chemiluminescent sandwich ELISA offers superior sensitivity and a wider dynamic range compared to traditional colorimetric assays . The following table outlines quantitative methods with their respective advantages:

For time-course experiments or drug response studies, it's critical to establish baseline phosphorylation and determine the linear detection range of the assay before experimental intervention. When comparing across multiple experimental conditions, include inter-assay calibrators on each blot or plate to allow normalization between experiments . For absolute quantification, consider phospho-peptide mass spectrometry approaches using heavy isotope-labeled standards corresponding to the Y1604 region.

How should researchers interpret discrepancies between different detection methods for phospho-ALK (Y1604)?

Discrepancies between detection methods for phospho-ALK (Y1604) require systematic analytical approaches to resolve. When Western blotting shows phosphorylation but IHC appears negative (or vice versa), researchers should consider several explanatory factors. First, epitope accessibility differs fundamentally between denatured proteins in Western blots versus fixed conformations in tissues. Sample preparation conditions critically affect phosphorylation preservation—Western blot samples typically undergo rapid denaturation that "locks in" phosphorylation states, while IHC fixation processes may allow dephosphorylation before epitope stabilization. Additionally, differences in antibody concentration between methods (1:1000 for WB versus 1:100-1:300 for IHC) can result in different detection thresholds.

To resolve such discrepancies, researchers should:

• Perform parallel validation using phosphatase-treated controls in both methods

• Assess method cross-validation using cell lines with known phospho-ALK status

• Consider the spatial heterogeneity in tissues that may be averaged out in Western blot lysates

• Evaluate the possibility of interfering factors specific to each method (e.g., endogenous phosphatases in IHC processing)

When alternative methods like ELISA contradict other results, evaluate the detection antibodies' epitopes—some ELISA kits use a capture antibody against phospho-ALK (Y1604) and a detection antibody against total ALK , which offers different specificity parameters than single-antibody methods. When discrepancies persist, orthogonal approaches like phospho-specific mass spectrometry may provide definitive assessment of phosphorylation status.

What are the appropriate strategies for normalizing phospho-ALK (Y1604) signals in quantitative analyses?

Appropriate normalization of phospho-ALK (Y1604) signals requires recognition that total ALK expression varies substantially between samples and experimental conditions. The scientifically valid normalization approach involves calculating the ratio of phospho-ALK (Y1604) to total ALK protein rather than normalizing to housekeeping proteins like β-actin or GAPDH. This approach specifically measures the proportion of ALK protein that is phosphorylated at Y1604, controlling for variations in total ALK expression that would otherwise confound interpretation.

When performing Western blot analyses, researchers should:

• Strip and reprobe membranes with total ALK antibody after phospho-ALK detection

• Use fluorescent secondary antibodies with distinct emission spectra for simultaneous detection

• Prepare technically replicated blots probed separately for phospho and total protein

For ELISA-based quantification, standard curves using recombinant phosphorylated ALK proteins at known concentrations enable absolute quantification rather than relative comparisons . In complex samples like tissue lysates, additional normalization to total protein concentration (determined by BCA or Bradford assay) prior to analysis ensures consistent loading. For longitudinal studies, inclusion of internal calibrator samples across experiments permits batch normalization to control for day-to-day technical variability. When analyzing fusion proteins like NPM-ALK (80 kDa) alongside full-length ALK (220 kDa), each form should be normalized to its corresponding total protein rather than combining signals .

How can researchers distinguish between specific ALK Y1604 phosphorylation and detection of other phosphorylated tyrosine kinases?

Distinguishing specific phospho-ALK (Y1604) detection from potential cross-reactivity with other phosphorylated tyrosine kinases requires rigorous specificity controls. The primary concern stems from the structural similarity of phosphorylated tyrosine residues across the kinome, particularly in conserved activation loop regions. To establish detection specificity, researchers should implement:

• Peptide competition assays comparing the specific phospho-Y1604 peptide immunogen (region 1570-1619) versus irrelevant phospho-tyrosine peptides

• Parallel analysis of ALK-knockout or ALK-depleted (siRNA) samples alongside wild-type samples

• Validation in cell models with ALK inhibitor treatment showing dose-dependent reduction in signal

• Analysis of purified recombinant tyrosine kinases to assess cross-reactivity profile

Phospho-ALK (Y1604) antibodies should ideally detect endogenous phospho-ALK at the correct molecular weight (220 kDa for full-length and 80 kDa for NPM-ALK) without significant bands at molecular weights corresponding to other tyrosine kinases. For reliable interpretation, antibody specificity data should include validation across multiple techniques (WB, IP, IHC, IF) as antibodies may show different cross-reactivity profiles in different applications . When questionable bands appear, confirmation with mass spectrometry analysis of immunoprecipitated proteins can definitively identify the phospho-proteins being detected. Importantly, recent antibody production methods using antiserum affinity-purified against the specific phosphopeptide immunogen substantially reduce cross-reactivity concerns compared to older generation antibodies .

What are the critical considerations when analyzing ALK Y1604 phosphorylation in clinical samples?

Analysis of ALK Y1604 phosphorylation in clinical samples presents unique challenges requiring specialized approaches. The pre-analytical phase is particularly critical—phosphorylation states begin degrading immediately upon sample collection due to endogenous phosphatase activity. Researchers should establish strict sample handling protocols with immediate flash-freezing or chemical fixation within minutes of collection. For surgical specimens, document cold ischemia time (time between removal and preservation) as this significantly impacts phospho-epitope integrity.

When working with formalin-fixed paraffin-embedded (FFPE) tissues:

• Optimize antigen retrieval conditions specifically for phospho-epitopes (typically EDTA-based buffers at pH 9.0)

• Include on-slide positive controls (ALK-positive cell lines) processed identically to patient samples

• Validate antibody performance on phosphatase-treated serial sections as negative controls

• Consider dual-staining for total ALK to identify phospho-negative/ALK-positive populations

For frozen clinical samples used in Western blotting or ELISA, standardized lysis procedures with robust phosphatase inhibition are essential . When comparing phosphorylation across patient cohorts (e.g., treatment-naïve versus resistant), batch processing minimizes technical variability. Heterogeneity within tumors necessitates multiple sampling from different regions when possible. Finally, clinical parameters including prior treatments, sample collection procedures, and time-to-processing should be incorporated into data interpretation, as these factors significantly influence phosphorylation status independent of biological differences.

What are the most common causes of weak or absent phospho-ALK (Y1604) signal in Western blotting?

Weak or absent phospho-ALK (Y1604) signal in Western blotting typically stems from several potential issues requiring systematic troubleshooting. Phospho-epitope degradation remains the most common cause—endogenous phosphatases rapidly dephosphorylate ALK unless immediately inhibited during sample preparation. Researchers should verify phosphatase inhibitor cocktail efficacy (must include both serine/threonine and tyrosine phosphatase inhibitors) and consider increasing sodium orthovanadate concentration to 2-5 mM if signal remains weak.

Other common technical issues include:

• Insufficient protein loading—phosphorylated ALK often represents a small fraction of total ALK; increase loading to 50-75 μg total protein

• Inappropriate antibody dilution—recommended 1:1000 dilution may require optimization to 1:500 for low abundance samples

• Inefficient protein transfer—large proteins like full-length ALK (220 kDa) transfer less efficiently; extend transfer time or use specialized high-molecular-weight transfer protocols

• Rapid signal decay—chemiluminescent detection systems vary in sensitivity; consider longer exposure times or higher-sensitivity substrates

Biological factors also contribute to weak signals: baseline ALK phosphorylation may be naturally low in certain cell types or conditions, requiring ALK activation (e.g., growth factor stimulation) prior to analysis. When ALK inhibitors have been applied to samples, even residual drug effect can suppress phosphorylation. Finally, confirm antibody compatibility with your sample species, as some phospho-ALK (Y1604) antibodies show human-specific reactivity while others detect human, mouse, and rat ALK .

How can researchers optimize immunohistochemistry protocols for detecting phospho-ALK (Y1604) in tissue sections?

Optimizing immunohistochemistry for phospho-ALK (Y1604) detection requires attention to each step from fixation through signal development. Phospho-epitopes are notoriously sensitive to standard histological processing. Begin with modified fixation—10% neutral buffered formalin supplemented with phosphatase inhibitors (1 mM sodium orthovanadate, 5 mM sodium fluoride) shows superior phospho-epitope preservation compared to standard fixatives. Limit fixation time to 12-24 hours to prevent overfixation that may mask epitopes.

For antigen retrieval, empirically test multiple conditions:

Block endogenous phosphatases using levamisole (for alkaline phosphatase detection systems) or additional sodium fluoride in blocking buffer. Antibody incubation should occur at 4°C overnight rather than at room temperature, with dilutions in the 1:100-1:300 range . Amplification systems such as tyramide signal amplification may enhance detection of low-abundance phospho-epitopes. Include serial section controls with phosphatase treatment to confirm phospho-specificity with each batch of staining. When conventional chromogenic IHC yields suboptimal results, consider fluorescent detection (IF) which often provides better signal-to-noise ratio for phospho-epitopes at 1:200-1:1000 dilutions .

What strategies can improve specificity when detecting phospho-ALK (Y1604) in complex biological samples?

Improving specificity for phospho-ALK (Y1604) detection in complex biological samples requires complementary approaches addressing both sample preparation and detection methodology. Immunoprecipitation (IP) prior to Western blotting significantly enhances specificity by enriching for ALK protein while removing potentially cross-reactive proteins. Using the phospho-ALK (Y1604) antibody for IP at a 1:50 dilution followed by Western blotting with total ALK antibody confirms that the immunoprecipitated protein is indeed ALK rather than a cross-reactive phospho-protein.

For enhanced specificity in direct detection methods:

• Implement dual epitope proximity assays (e.g., Proximity Ligation Assay) using antibodies against phospho-Y1604 and a distinct ALK epitope, generating signal only when both epitopes are in close proximity

• Perform sequential probing with phospho-specific and total protein antibodies with different visualization methods

• Include competition controls with phospho-peptide (1570-1619 region) and non-phospho-peptide to demonstrate phospho-specificity

• Treat parallel samples with lambda phosphatase to establish baseline non-phosphorylated signal

In mass spectrometry-based approaches, enrich for phospho-peptides using titanium dioxide or immobilized metal affinity chromatography prior to analysis. For clinical samples with high background, consider using ELISA-based detection which offers improved specificity through the sandwich format with capture antibodies against phospho-ALK (Y1604) and detection antibodies against total ALK . This approach ensures that both epitopes must be present on the same protein for signal generation, substantially reducing false positives from cross-reactivity.

How should researchers address batch-to-batch variability in phospho-ALK (Y1604) antibody performance?

Addressing batch-to-batch variability in phospho-ALK (Y1604) antibody performance requires proactive validation strategies and standardized experimental approaches. When receiving a new antibody lot, perform direct comparison with the previous lot on identical samples using consistent protocols. Establish a panel of reference samples with known phospho-ALK (Y1604) status—ideally including positive controls (ALK-expressing cell lines), negative controls (ALK-negative cell lines), and dose-response samples (ALK-positive cells treated with varying concentrations of ALK inhibitors).

To systematically evaluate lot consistency:

• Compare signal intensity at standard dilution (1:1000 for WB , 1:100-1:300 for IHC )

• Assess signal-to-noise ratio using background measurements in negative control samples

• Determine effective working dilution range for each application

• Document EC50 values in dose-response experiments with ALK inhibitors

When significant variability is detected, consider creating a master calibration curve for each lot to normalize experimental data. For critical long-term studies, reserve sufficient antibody from a single lot to complete the entire experimental series. Alternatively, create a standard reference sample set that can be run alongside experimental samples to allow batch correction during data analysis. When antibody production method information is available, polyclonal antibodies purified through affinity chromatography using epitope-specific immunogen typically show better lot-to-lot consistency than those purified by protein A/G methods alone.

How can phospho-ALK (Y1604) antibodies be integrated into multiplexed detection systems?

Integration of phospho-ALK (Y1604) detection into multiplexed systems enables simultaneous analysis of ALK phosphorylation alongside other signaling nodes. Mass cytometry (CyTOF) represents a powerful platform where metal-conjugated phospho-ALK (Y1604) antibodies can be combined with dozens of other signaling markers without spectral overlap limitations. For microscopy-based multiplexing, sequential immunofluorescence with iterative antibody stripping or multi-epitope ligand cartography (MELC) allows co-detection of phospho-ALK (Y1604) with downstream effectors like phospho-ERK or phospho-PLCγ.

Emerging multiplexed methods with phospho-ALK (Y1604) antibodies include:

• Digital spatial profiling combining phospho-ALK detection with transcriptomic analysis in spatially resolved tissue regions

• Antibody-barcode conjugates for high-plex digital quantification of phospho-ALK alongside hundreds of other proteins

• Microfluidic-based single-cell Western blotting detecting phospho-ALK (Y1604) and total ALK in individual cells

• Luminex bead-based assays for simultaneous quantification of multiple phosphorylation sites on ALK

• Proximity extension assays combining phospho-ALK antibody specificity with PCR sensitivity

For optimal multiplexed detection, antibody selection requires careful consideration of cross-reactivity profiles and compatible secondary detection systems. ELISA-based platforms can be adapted to multiplex format using chemiluminescent detection combined with spatial separation of capture antibodies. When implementing any multiplexed system, validation should confirm that detection of phospho-ALK (Y1604) remains specific and quantitative within the multiplexed format compared to single-plex detection.

What is the role of phospho-ALK (Y1604) antibodies in studying ALK inhibitor resistance mechanisms?

Phospho-ALK (Y1604) antibodies serve as critical tools for investigating resistance mechanisms to ALK inhibitors in research and clinical settings. Since Y1604 phosphorylation is essential for oncogenic signaling and PLCγ interaction , persistent phosphorylation despite inhibitor treatment provides direct evidence of drug resistance. Researchers can employ these antibodies to characterize multiple resistance mechanisms, including:

• On-target resistance—secondary mutations in ALK kinase domain that prevent inhibitor binding while maintaining catalytic activity

• Bypass track activation—alternative signaling pathways compensating for ALK inhibition

• Pharmacokinetic resistance—insufficient drug exposure at tumor site

• ALK amplification—increased ALK protein expression overwhelming inhibitor concentration

In patient-derived xenograft models, longitudinal monitoring of Y1604 phosphorylation during treatment reveals the emergence of resistant clones before macroscopic progression. Cell line models with induced resistance can be characterized using phospho-ALK (Y1604) detection by Western blotting (1:1000 dilution) or ELISA to quantify residual ALK activity. Phospho-ALK (Y1604) immunohistochemistry (1:100-1:300 dilution) on serial biopsies from patients undergoing ALK inhibitor therapy provides crucial information about in vivo drug efficacy and resistance evolution. By correlating phosphorylation status with genomic profiling of resistance mutations, researchers can develop rational combinations targeting specific resistance mechanisms, positioning phospho-ALK (Y1604) antibodies as essential companions in precision medicine approaches to ALK-driven malignancies.

How are phospho-ALK (Y1604) antibodies utilized in high-throughput drug discovery platforms?

Phospho-ALK (Y1604) antibodies have become instrumental in high-throughput drug discovery platforms targeting ALK-dependent cancers. The chemiluminescent sandwich ELISA format offers particular advantages for screening applications, with its wide dynamic range and small sample requirements enabling miniaturization to 384-well or higher density formats . In automated screening platforms, cells expressing either full-length ALK or oncogenic fusion proteins (NPM-ALK, EML4-ALK) are treated with compound libraries, followed by in-cell detection of Y1604 phosphorylation status.

Key implementations in drug discovery include:

• Primary screens identifying compounds that inhibit ALK phosphorylation at Y1604

• Secondary assays characterizing potency (IC50) against wild-type and mutant ALK variants

• Selectivity profiling against panels of kinase domain mutations associated with inhibitor resistance

• Mechanism-of-action studies distinguishing between direct ALK inhibitors versus modulators of upstream regulatory pathways

• In vivo pharmacodynamic biomarker assessment in preclinical models

Homogeneous assay formats like AlphaLISA or time-resolved FRET using phospho-ALK (Y1604) antibodies paired with total ALK detection enable true high-throughput applications without wash steps. For more complex screening paradigms, phospho-ALK (Y1604) detection can be combined with cell viability or apoptosis readouts in multiplexed high-content imaging platforms. The relative phosphorylation level (phospho-ALK/total ALK ratio) provides a robust normalization method accounting for variations in ALK expression between cell lines or primary samples, resulting in more reliable hit identification .

What advances in phospho-ALK (Y1604) detection are enabling single-cell analysis of ALK signaling heterogeneity?

Recent technological advances in phospho-ALK (Y1604) detection are revealing previously unappreciated heterogeneity in ALK signaling at the single-cell level. Mass cytometry (CyTOF) with metal-conjugated phospho-ALK (Y1604) antibodies enables high-dimensional analysis of ALK phosphorylation simultaneously with dozens of other signaling proteins, cell surface markers, and transcription factors at single-cell resolution. This approach has revealed distinct cell subpopulations with differential ALK activation states within seemingly homogeneous tumors.

Emerging single-cell technologies for phospho-ALK (Y1604) analysis include:

• Microfluidic single-cell Western blotting detecting phospho-ALK (Y1604) and total ALK in individual cells with retention of morphological information

• Imaging mass cytometry (IMC) mapping spatial distribution of phospho-ALK in tissue contexts with subcellular resolution

• Single-cell phospho-proteomics isolating individual cells for mass spectrometry analysis of Y1604 phosphorylation

• Digital spatial profiling quantifying phospho-ALK in spatially defined tissue regions at near-single-cell resolution

• Live-cell reporters based on phospho-specific antibody fragments monitoring Y1604 phosphorylation dynamics in real-time

These approaches reveal that ALK inhibitor response is rarely uniform across all cells in a population, with resistant subpopulations often detectable before clinical progression. The gold standard for antibody-based single-cell studies remains careful validation of phospho-specificity (1:200-1:1000 dilution range for immunofluorescence) , particularly important at the sensitivity levels required for single-cell analysis. Integration of phospho-ALK (Y1604) single-cell data with genomic and transcriptomic analyses from the same samples is providing unprecedented insights into the relationship between ALK genetic alterations, phosphorylation states, and downstream pathway activation in individual cells.