PYD2 Antibody

What is Phospho-PYK2/FAK2 (Y402) Antibody and what does it detect?

Phospho-PYK2/FAK2 (Y402) Antibody specifically recognizes the phosphorylated tyrosine residue at position 402 of Protein Tyrosine Kinase 2 (PYK2), also known as Focal Adhesion Kinase 2 (FAK2). This antibody detects activated PYK2, which plays critical roles in cellular signaling pathways .

The antibody has been validated for detecting specific bands at approximately 105-115 kDa in Western blot applications and approximately 113 kDa in Simple Western systems when working with human cell lines . This specificity makes it particularly valuable for studying signaling cascades, especially in immune cells as demonstrated by its application in detecting phosphorylated PYK2 in Raji human Burkitt's lymphoma and Jurkat human acute T cell leukemia cell lines .

What validated applications exist for Phospho-PYK2/FAK2 (Y402) Antibody?

Based on research data and user reviews, Phospho-PYK2/FAK2 (Y402) Antibody has been validated for multiple applications as shown in the following table:

Researchers should note that optimal dilutions should be determined for each specific application and laboratory condition .

How should PYK2 antibody specificity be validated before experimental use?

When validating PYK2 antibody specificity, researchers should implement a multi-step approach:

• Positive control verification: Test the antibody on samples known to express phosphorylated PYK2, such as pervanadate-treated Raji or Jurkat cell lines .

• Negative control testing: Include untreated cells where phosphorylation is minimal as comparative controls .

• Stimulation response: Confirm increased signal after treatments that activate PYK2 signaling (e.g., pervanadate treatment or CD3e antibody stimulation as demonstrated in published protocols) .

• Molecular weight verification: Confirm detection at the expected molecular weight (105-115 kDa for PYK2) .

• Cross-reactivity assessment: Test against closely related proteins like FAK1 to ensure specificity for the intended target.

How should cell stimulation protocols be optimized when studying PYK2 phosphorylation?

Optimization of cell stimulation protocols for PYK2 phosphorylation studies should consider:

Stimulation parameters:

• Pervanadate concentration: Typically effective at 0.2-1.0 mM for 30 minutes

• Receptor-mediated activation: For T cells, anti-CD3e antibody at 10 μg/mL for 15 minutes has proven effective

• Time course: Monitor phosphorylation at multiple time points (5, 15, 30, 60 minutes) to identify peak activation

Buffer considerations:

• Use phosphatase inhibitors to prevent dephosphorylation during sample preparation

• Process samples quickly at cold temperatures to preserve phosphorylation state

• Select appropriate lysis buffers that maintain protein conformation while effectively solubilizing membrane-associated proteins

Validation controls:

• Include both untreated and treated samples from the same cell population

• Consider using siRNA or CRISPR knockout cells for antibody specificity validation

What are the optimal detection methods for quantifying PYK2 phosphorylation in different sample types?

The selection of method should consider the research question, required sensitivity, and available sample quantity. For low abundance samples, Simple Western or ELISA may offer superior sensitivity compared to traditional Western blotting .

How can PYK2 antibodies be incorporated into structural biology approaches for understanding signaling complexes?

Integrating PYK2 antibodies into structural biology requires sophisticated approaches similar to those used for other antibody-antigen structural studies:

• Epitope mapping techniques:

  • X-ray crystallography of antibody-RBD complexes can reveal precise interaction sites, as demonstrated for other antibody-target interactions

  • High-resolution cryo-electron microscopy (cryo-EM) can visualize antibody binding to larger protein complexes, potentially useful for PYK2 complexes with binding partners

• Conformational state detection:

  • Different antibodies may recognize distinct conformational states of PYK2 (similar to how some antibodies recognize "up" vs "down" conformations of targets)

  • This property can be exploited to "lock" PYK2 in specific confirmations for structural studies

• Methodological considerations:

  • Generation of Fab fragments to reduce size and flexibility for structural studies

  • Co-crystallization optimization through screening various buffer conditions

  • Use of computational modeling to predict and analyze binding modes before experimental validation

What strategies can resolve data contradictions when different PYK2 phospho-antibodies yield conflicting results?

When facing contradictory results from different phospho-specific PYK2 antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope characterization:

  • Determine precise epitope recognition sites for each antibody

  • Consider that antibodies targeting different regions around the phosphorylation site may have varying accessibility depending on protein conformation or binding partners

• Validation with orthogonal techniques:

  • Employ mass spectrometry to definitively identify phosphorylation sites

  • Use phosphatase treatment controls to confirm signal specificity

  • Validate with genetic approaches (phospho-null mutants)

• Binding affinity assessment:

  • Compare affinity constants of different antibodies

  • Investigate potential cross-reactivity with closely related epitopes

• Data integration framework:

  • Develop a weighted scoring system considering antibody validation extent

  • Implement multivariate analysis to identify patterns in seemingly contradictory results

  • Consider contextual factors (cell type, stimulation protocol, detection method) that might explain divergent findings

How can machine learning approaches enhance PYK2 antibody specificity and cross-reactivity profiling?

Advanced computational approaches can significantly improve antibody specificity profiling:

• Biophysics-informed modeling:

  • Models can be trained on experimental antibody selection data to identify distinct binding modes associated with specific ligands

  • This approach enables prediction of antibody-epitope interactions beyond those observed experimentally

• Specificity engineering:

  • Computational design of antibodies with customized specificity profiles can be achieved through the identification of key binding determinants

  • These models can disentangle multiple binding modes associated with specific ligands, even when they are chemically very similar

• Experimental validation:

  • High-throughput sequencing coupled with computational analysis allows validation of specificity predictions

  • Phage display experiments with diverse combinations of closely related ligands can generate training data for computational models

• Applications to PYK2 antibodies:

  • Models could predict cross-reactivity with related kinases or phosphorylation sites

  • Enable design of antibodies that specifically discriminate between different phosphorylation states or conformations of PYK2

What are the critical parameters for optimizing multiplex detection systems incorporating PYK2 phosphorylation?

When developing multiplex systems that include PYK2 phosphorylation detection:

• Antibody compatibility assessment:

  • Test for antibody cross-reactivity and interference

  • Validate signal specificity in the presence of multiple detection reagents

  • Optimize antibody concentrations to achieve balanced sensitivity across targets

• Signal normalization strategy:

  • Implement housekeeping proteins as internal controls

  • Develop standard curves for each target in multiplex format

  • Consider ratiometric measurements (phospho-PYK2/total PYK2)

• Technical parameters:

  • Optimize incubation times and washing protocols specific to multiplex formats

  • Select compatible fluorophores or tags with minimal spectral overlap

  • Validate with both positive and negative control samples

• Data analysis frameworks:

  • Develop algorithms for correcting signal spillover between channels

  • Implement statistical approaches for handling multiparameter data

  • Consider machine learning for pattern recognition in complex datasets

How might next-generation antibody engineering improve PYK2 phosphorylation detection sensitivity and specificity?

Emerging antibody engineering approaches hold promise for enhanced PYK2 detection:

• Stereotypic antibody development:

  • Leveraging knowledge of stereotypic immunoglobulin gene arrangements shared among multiple individuals

  • Engineering conserved features like di-cysteine motifs within CDR-H3 regions that confer high-affinity binding

• Structure-guided optimization:

  • Using high-resolution structural data to modify CDR loops for improved specificity

  • Engineering heavy chain-only recognition systems for accessing cryptic epitopes

• Antibody cocktail approaches:

  • Developing complementary antibody combinations that recognize distinct epitopes

  • Creating synergistic binding profiles that enhance signal-to-noise ratios

• Novel modalities:

  • Engineered nanobodies with enhanced tissue penetration

  • Synthetic binding proteins based on alternative scaffolds

  • Proximity-dependent labeling antibodies for detecting PYK2 in specific protein complexes

What are the emerging applications for PYK2 antibodies in single-cell phospho-signaling analysis?

PYK2 antibody applications are expanding into single-cell analysis through:

• Technical innovations:

  • Integration with mass cytometry (CyTOF) for high-dimensional analysis of phospho-signaling

  • Compatibility with microfluidic platforms for dynamic stimulation studies

  • Development of specialized fixation protocols that preserve phosphorylation states while enabling single-cell resolution

• Analytical approaches:

  • Trajectory analysis of phosphorylation events at the single-cell level

  • Correlation of PYK2 phosphorylation with other signaling nodes

  • Identification of rare cell populations with distinctive PYK2 activation profiles

• Biological insights:

  • Mapping cell-type-specific PYK2 signaling networks

  • Identifying signaling heterogeneity within seemingly homogeneous populations

  • Connecting PYK2 activation patterns to functional cellular outcomes