NCK2 Antibody

Basic Research Questions

• What is NCK2 and what cellular functions does it perform?

  NCK2 (also known as NCK adaptor protein 2, NCKbeta, or GRB4) is a 42.9 kDa adaptor protein comprised of three N-terminal SH3 domains and one C-terminal SH2 domain . It functions as a critical signaling molecule that associates with tyrosine-phosphorylated growth factor receptors or their cellular substrates . NCK2 is ubiquitously expressed across many tissue types with subcellular localization primarily in the endoplasmic reticulum and cytoplasm .

  The protein plays essential roles in:

  • Regulating cytoskeletal dynamics and organization

  • Mediating cell migration and adhesion processes

  • Coupling tyrosine kinase signaling pathways (like EGF and PDGF) to downstream effectors

  • Controlling cell proliferation via inhibition of growth factor-induced DNA synthesis

  • Regulating adipogenesis and metabolic homeostasis

  Methodologically, researchers can study NCK2 function through knockout/knockdown approaches, overexpression systems, and by examining its interactions with binding partners using co-immunoprecipitation or proximity ligation assays.

• What are the most common applications for NCK2 antibodies in research?

  NCK2 antibodies are utilized across multiple experimental techniques, with the following applications being most common:

  Application	Typical Dilution	Sample Types	Key Considerations
  Western Blot (WB)	1:500-1:2000	Cell/tissue lysates	Expected band size: 43 kDa
  Immunohistochemistry (IHC-P)	1:50-1:200	FFPE tissue sections	May require antigen retrieval with TE buffer pH 9.0
  Immunofluorescence (IF)	1:50-1:500	Fixed cells	Useful for subcellular localization studies
  Immunoprecipitation (IP)	Variable	Cell lysates	Effective for protein-protein interaction studies
  ELISA	1:500-1:5000	Purified protein, serum	Quantitative detection method

  For optimal results, researchers should validate each antibody for their specific application and sample type, as reactivity can vary between antibodies and experimental conditions .

• How can I differentiate between NCK1 and NCK2 in my experiments?

  Despite sharing a high degree of amino acid identity and similar domain structures (both ~47 kDa), NCK1 and NCK2 can be distinguished through several methodological approaches:

  • Selective antibodies: Use antibodies specifically raised against unique epitopes of NCK2 (or NCK1). For example, antibody clones like 8.8 specifically target NCK2 .

  • Western blotting: Although similar in size, subtle mobility differences may be detected with high-resolution SDS-PAGE.

  • qRT-PCR: Design primers specific to unique regions of the NCK2 gene to distinguish its expression from NCK1.

  • Functional assays: NCK2, but not NCK1, is involved in specific processes like axon trajectory during limb development and acts as a positive regulator of Erk pathway stimulation via T cell antigen receptor .

  • Expression pattern analysis: While both are widely expressed, tissue-specific differences in expression ratios can help identify the predominant isoform.

  When investigating phenotypes, using isoform-specific knockdown approaches (siRNA/shRNA) that target unique regions can help determine the specific contributions of NCK2 versus NCK1 .

• What controls should I include when working with NCK2 antibodies?

  Proper controls are essential for ensuring experimental validity when working with NCK2 antibodies:

  • Positive control: Use cell lines or tissues known to express NCK2 (e.g., U-937 cells, thymus tissue, or human placenta) .

  • Negative control: Include samples where NCK2 is known to be absent or depleted, such as:

    • NCK2 knockout/knockdown cells (CRISPR or siRNA-treated)

    • Isotype control antibodies that match the host species and immunoglobulin class

  • Peptide competition/blocking: Pre-incubate the antibody with the immunizing peptide to confirm specificity.

  • Loading controls: For Western blots, include housekeeping proteins (e.g., GAPDH, β-actin) to normalize for loading variations.

  • Secondary antibody-only controls: To detect non-specific binding of the secondary antibody.

  For immunocytochemistry/immunohistochemistry, include both primary antibody omission controls and isotype controls to distinguish between specific and non-specific binding .

Advanced Research Questions

• How does NCK2 regulate cytoskeletal dynamics and cell migration at the molecular level?

  NCK2 functions as a critical mediator between receptor tyrosine kinases and cytoskeletal organization, using its modular architecture to orchestrate complex molecular interactions:

  • PDGFR-β signaling: NCK2 binds to phosphorylated tyrosine 1009 on PDGFR-β. Experimental evidence shows that NCK2 overexpression inhibits PDGF-induced membrane ruffling and lamellipod formation, directly impacting cell motility and morphology .

  • PAK interaction pathway: Through its SH3 domains, NCK2 binds to proline-rich domains on PAK (p21-activated kinase), a known regulator of actin cytoskeleton . This interaction can be studied using domain-specific mutations in the SH3 domains followed by co-immunoprecipitation assays.

  • Temporal dynamics: Real-time imaging using fluorescent-tagged NCK2 constructs reveals its recruitment to focal adhesions during cell migration, allowing for quantitative analysis of its temporal regulation.

  • Quantitative phenotype analysis: Researchers should employ multiple readouts when assessing migration phenotypes, including:

    • Scratch wound healing assays (measuring closure rate)

    • Transwell migration assays (counting cells traversing the membrane)

    • Single-cell tracking (analyzing velocity and directionality)

    • Kymograph analysis (evaluating protrusion dynamics)

  When examining NCK2's role in cytoskeletal reorganization, it is essential to differentiate between direct effects on actin polymerization versus indirect effects through other signaling proteins by using domain-specific mutants of NCK2 in rescue experiments .

• What methodological approaches should be used to study NCK2's role in adipogenesis and metabolic regulation?

  Recent research has identified NCK2 as an unexpected regulator of adipogenesis and metabolism. The following methodological approaches are recommended for investigating this function:

  • Expression profiling during differentiation: Monitor NCK2 expression levels during adipocyte differentiation using qRT-PCR, Western blotting, and immunofluorescence. Data indicates NCK2 is upregulated during differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes .

  • Loss-of-function studies: Use NCK2 knockout mice or siRNA-mediated knockdown in preadipocyte cell lines, followed by:

    • Oil Red O staining to quantify lipid accumulation

    • mRNA analysis of adipocyte markers (PPARγ, C/EBPα, FABP4)

    • Glucose uptake assays to assess metabolic function

    • Lipolysis assays to measure triglyceride breakdown

  • Gain-of-function approaches: Overexpress NCK2 in preadipocytes and assess:

    • Differentiation capacity

    • PPARγ nuclear translocation using fractionation and immunofluorescence

    • Activation of adipogenic signaling pathways

  • Mechanistic investigations: Examine potential interaction between NCK2 and PPARγ or Dok1 using:

    • Co-immunoprecipitation under endogenous expression conditions

    • Proximity ligation assays in intact cells

    • Domain mapping to identify interaction regions

  In human studies, researchers should examine NCK2 expression in adipose tissue biopsies from individuals with different BMI levels, as NCK2 has been found downregulated in severely obese individuals' adipose tissue .

• How can researchers effectively investigate the relationship between NCK2 and receptor tyrosine kinase signaling pathways?

  Studying NCK2's role in receptor tyrosine kinase (RTK) signaling requires multifaceted approaches:

  • SH2 domain interaction analysis: The SH2 domain of NCK2 mediates binding to phosphotyrosine residues on activated RTKs and their substrates. Researchers should employ:

    • Phosphotyrosine-specific pulldowns using recombinant SH2 domain

    • Phosphoproteomic analysis to identify NCK2 binding partners after RTK stimulation

    • Mutagenesis of the SH2 domain to abolish phosphotyrosine binding

  • Pathway-specific readouts: Measure downstream signaling after RTK activation in the presence/absence of NCK2:

    • MAPK/ERK activation (phospho-specific antibodies)

    • PI3K/AKT signaling (phospho-AKT detection)

    • STAT pathway activation

  • Spatiotemporal dynamics: Use live-cell imaging with fluorescently-tagged NCK2 to observe:

    • Recruitment kinetics to activated receptors

    • Co-localization with endocytic markers

    • Redistribution upon RTK activation

  • Functional output assays: The SH2 domain of NCK2 has been implicated in inhibiting EGF- and PDGF-induced DNA synthesis, highlighting its role in controlling cell proliferation . Measure:

    • BrdU incorporation

    • EdU labeling

    • Cell cycle progression

    • Colony formation

  When investigating isoform-specific functions, combine NCK2 knockdown with re-expression of either NCK1 or NCK2 to determine whether one isoform can compensate for the other's functions in RTK signaling .

• What are the experimental considerations when studying NCK2's role in T cell receptor signaling and activation?

  NCK2 plays a distinct role in T cell receptor (TCR) signaling that differs from NCK1, requiring specific experimental approaches:

  • T cell activation models: Use Jurkat T cells or primary T cells with:

    • Anti-CD3/CD28 stimulation

    • Antigen-presenting cell co-culture

    • Pharmacological activators (PMA/ionomycin as controls)

  • Quantitative signaling readouts: As NCK2 specifically regulates Erk pathway stimulation via TCR , measure:

    • Phospho-Erk levels by Western blot or flow cytometry

    • Calcium flux using Fluo-4 or Indo-1 dyes

    • Nuclear translocation of NFAT and NF-κB

    • CD69 and IL-2 expression as functional readouts

  • NCK1 vs. NCK2 comparison: Design experiments that can distinguish their roles:

    • Isoform-specific knockdown/knockout

    • Structure-function analysis with chimeric proteins

    • Domain-swapping experiments

  • Interaction partners: Identify TCR-specific binding partners of NCK2 through:

    • Immunoprecipitation following TCR engagement

    • Proximity-based labeling (BioID or APEX)

    • Mass spectrometry analysis of NCK2 complexes

  When interpreting results, researchers should consider the activation state of T cells, as NCK2's function may differ between resting and activated states, or between different T cell subsets .

• How does post-translational modification of NCK2 affect its function and experimental detection?

  NCK2 undergoes several post-translational modifications (PTMs) that regulate its function and may affect antibody recognition:

  • Phosphorylation: NCK2 is subject to phosphorylation , which may alter its:

    • Binding affinity for interaction partners

    • Subcellular localization

    • Stability or turnover

  Methodological approaches to study phosphorylation include:

  • Phospho-specific antibodies (if available)

  • Phos-tag SDS-PAGE to separate phosphorylated forms

  • Mass spectrometry to map phosphorylation sites

  • Phosphatase treatment of samples to verify phosphorylation

  • Experimental considerations: When detecting NCK2, researchers should be aware that:

    • Different antibodies may have varying sensitivity to phosphorylated forms

    • Sample preparation methods may preserve or destroy phosphorylation

    • Phosphorylation status may change rapidly during cell lysis

  • Other potential PTMs: Based on similar adaptor proteins, researchers should consider:

    • Ubiquitination (affecting stability)

    • SUMOylation (altering localization or interactions)

    • Acetylation (potentially affecting domain accessibility)

  When studying PTMs, combining immunoprecipitation with specific PTM detection methods provides the most comprehensive analysis of NCK2 modification states in different cellular contexts .

• What strategies can researchers employ to study NCK2's potential as a therapeutic target in cancer or metabolic diseases?

  Given NCK2's implications in cancer progression (melanoma, breast cancer) and metabolic regulation, several approaches can evaluate its therapeutic potential:

  • Expression correlation analysis: Compare NCK2 levels between:

    • Metastatic vs. non-metastatic melanoma samples

    • Different breast cancer subtypes

    • Adipose tissue from lean vs. obese subjects

  • Functional inhibition approaches:

    • Domain-specific peptide inhibitors targeting SH2 or SH3 domains

    • Small molecule inhibitors of protein-protein interactions

    • Structure-based drug design targeting NCK2's critical interfaces

  • Phenotypic rescue experiments:

    • Determine if NCK2 restoration in downregulated contexts reverses pathological phenotypes

    • Assess whether domain-specific mutants can partially rescue function

  • Combination therapy models:

    • Test NCK2 inhibition alongside established cancer therapies

    • Evaluate NCK2 modulation with metabolic disease treatments

  • Biomarker potential assessment:

    • Correlate NCK2 levels with disease progression

    • Develop sensitive detection methods for tissue or liquid biopsies

  Researchers should employ both in vitro cell models and in vivo systems (xenografts, genetic mouse models) to comprehensively evaluate the therapeutic implications of targeting NCK2, while considering potential compensatory mechanisms by NCK1 .

Technical Considerations

• What factors should be considered when selecting an NCK2 antibody for specific applications?

  Selecting the appropriate NCK2 antibody requires evaluation of several critical factors:

  Factor	Considerations	Practical Tips
  Epitope	Target region of NCK2 (N-terminal, SH3 domains, SH2 domain, etc.)	Choose antibodies targeting conserved regions for cross-species applications
  Host Species	Rabbit, mouse, etc.	Consider compatibility with other antibodies for co-staining experiments
  Clonality	Monoclonal vs. polyclonal	Monoclonals offer higher specificity; polyclonals may provide stronger signals
  Validated Applications	WB, IHC, IF, IP, ELISA	Verify the antibody has been validated for your specific application
  Species Reactivity	Human, mouse, rat, etc.	Many NCK2 antibodies react with multiple species due to sequence conservation
  Format	Unconjugated, HRP/AP conjugated, fluorescently labeled	Select based on detection method and experimental design

  Researchers should review validation data (Western blot images, IHC staining patterns) and citations in the literature before selecting an antibody, particularly noting whether the antibody detects endogenous levels of NCK2 rather than just overexpressed protein .

• How should sample preparation be optimized for NCK2 detection in different experimental contexts?

  Proper sample preparation is critical for successful NCK2 detection:

  • For Western blotting:

    • Use lysis buffers containing phosphatase inhibitors to preserve phosphorylation status

    • Include protease inhibitors to prevent degradation

    • Consider subcellular fractionation for analyzing distribution between cytoplasm and membrane

    • Expected molecular weight is approximately 43 kDa

  • For immunohistochemistry:

    • Optimal fixation: 10% neutral buffered formalin

    • Antigen retrieval: TE buffer pH 9.0 is often recommended

    • Alternative method: Citrate buffer pH 6.0

    • Blocking: Use 5-10% normal serum matching the secondary antibody host

  • For immunofluorescence:

    • Fixation: 4% paraformaldehyde preserves structure

    • Permeabilization: 0.1-0.5% Triton X-100 or 0.1% saponin

    • Consider co-staining with markers for ER, cytoskeleton, or other binding partners

  • For immunoprecipitation:

    • Use mild lysis conditions to preserve protein-protein interactions

    • Consider crosslinking if interactions are transient

    • Include appropriate controls (IgG, pre-immune serum)

  When investigating NCK2's role in dynamic processes like receptor signaling or cytoskeletal remodeling, timing of sample collection is crucial—consider collecting multiple time points after stimulation .