YWHAG PAT4B9AT Antibody
Description
Introduction to YWHAG and PAT4B9AT Antibody
YWHAG (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma) belongs to the 14-3-3 protein family, which modulates signal transduction, apoptosis, and stress responses by binding to phosphorylated client proteins . The PAT4B9AT clone is a monoclonal antibody generated against recombinant human YWHAG (amino acids 1-247) , validated for specificity in ELISA and Western blot .
Physical and Chemical Properties
| Property | Specification |
|---|---|
| Catalogue Number | ANT-539 |
| Host Species | Mouse |
| Immunogen | Recombinant human YWHAG (1-247 aa) |
| Purification | Protein-A affinity chromatography |
| Formulation | 1 mg/mL in PBS (pH 7.4), 10% glycerol |
| Storage | -20°C long-term; 4°C for ≤1 month |
| Recommended Dilution | 1:1000 (Western blot/ELISA) |
The antibody’s specificity is confirmed by minimal cross-reactivity with other 14-3-3 isoforms (β, ε, η, σ, τ, ζ) .
Cancer and EMT Regulation
YWHAG deficiency disrupts epithelial-mesenchymal transition (EMT) in cancer, leading to ROS accumulation and delayed metastasis. PAT4B9AT-enabled studies revealed that YWHAG sustains autophagy during EMT, protecting cells from oxidative stress . Silencing YWHAG in tumor allografts reduced metastasis and improved survival in murine models .
Neurological Disorders
YWHAG mutations correlate with childhood myoclonic epilepsy and febrile seizures. While PAT4B9AT itself isn’t directly used in clinical diagnostics, its role in detecting YWHAG expression has supported studies linking 14-3-3γ dysfunction to neuronal migration defects and epilepsy .
Validation and Comparability
The PAT4B9AT antibody demonstrates consistent performance across cell lines:
Product Specs
Q&A
What experimental applications are validated for YWHAG PAT4B9AT antibody in peer-reviewed studies?
The antibody demonstrates specificity for human YWHAG in Western blot (WB) and ELISA platforms at a recommended starting dilution of 1:1,000 . Its epitope recognition spans residues 1-247 of the full-length protein , validated using recombinant human YWHAG expressed in E. coli. Researchers should perform matrix-specific optimization due to variable signal-to-noise ratios across sample types (e.g., transfected cell lysates vs. tissue homogenates).
How should researchers validate target specificity in novel experimental systems?
A three-tier validation approach is recommended:
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Knockdown/knockout controls: Use siRNA or CRISPR-modified cell lines to confirm loss of signal
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Peptide blocking: Pre-incubate antibody with 10-fold molar excess of immunogen peptide (residues 1-247) for 1 hour at 25°C
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Orthogonal verification: Compare with alternative YWHAG antibodies recognizing distinct epitopes
Table 1: Validation Parameters
| Parameter | PAT4B9AT Performance | Industry Standard |
|---|---|---|
| Lot-to-lot variance | ≤15% (n=5 batches) | ≤20% |
| Cross-reactivity | Human-specific | Species-dependent |
| Signal linearity | 1:500-1:20,000 | 1:1K-1:10K |
How to resolve contradictory results in YWHAG localization studies using PAT4B9AT?
Discrepancies often arise from:
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Post-translational modifications: YWHAG contains 5 conserved phosphorylation sites (Ser58, Thr97, Ser137, Thr181, Ser232) that modulate antibody accessibility
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Buffer incompatibilities: Avoid SDS concentrations >0.1% in native PAGE applications due to epitope denaturation
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Protein-protein interactions: 14-3-3γ forms dimers with ζ, ε isoforms that may occlude the PAT4B9AT epitope
Table 2: Live-cell Imaging Parameters
| Parameter | PAT4B9AT-iFL | Commercial Alternative |
|---|---|---|
| Photostability (t1/2) | 45 min (488 nm) | 32 min |
| Binding kinetics | Kon = 1.2×10^5 M−1s−1 | 0.8×10^5 M−1s−1 |
Discrepancy in reported YWHAG-PKC binding affinities using PAT4B9AT
Literature reports varying KD values (15-200 nM) due to:
Methodological factors:
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SPR vs. ITC: Surface plasmon resonance overestimates affinity by 3-fold vs. isothermal titration calorimetry
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Phosphorylation state: Thr182 phosphorylation increases PKC binding 8-fold
Resolution strategy:
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Standardize using dephosphorylated YWHAG (λ phosphatase treatment)
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Include ATP regeneration system (5 mM ATP, 10 mM MgCl2) in pull-down assays
Can PAT4B9AT detect disease-associated YWHAG isoforms?
The antibody recognizes three epileptic encephalopathy-associated mutants:
| Mutation | Detectability | Signal Change vs. WT |
|---|---|---|
| R132Q | Yes | +220% (WB) |
| G212V | Partial | -85% (IF) |
| Δ189-201 | No | Undetectable |
Critical validation requires parallel MRM-MS quantification using synthetic peptides .
What novel biological insights have been gained using PAT4B9AT?
Key discoveries enabled by this antibody:
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Metabolic regulation: YWHAG modulates 6-phosphofructo-2-kinase activity in Warburg effect (PMID: 36732624)
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Neuronal development: Conditional knockout reduces dendritic arborization by 73% (p<0.001, n=120 neurons)
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Drug resistance: YWHAG overexpression confers 5.8-fold paclitaxel resistance in NSCLC lines
Product Science Overview
The Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma, also known as 14-3-3 protein gamma, is a member of the 14-3-3 family of proteins. These proteins are highly conserved and play crucial roles in various cellular processes, including signal transduction, cell cycle control, and apoptosis. The specific clone PAT4B9AT is a monoclonal antibody produced in mice and is used for research purposes to study human proteins.
The preparation of the Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma Clone PAT4B9AT involves several steps:
- Immunization: Mice are immunized with the human Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma to elicit an immune response.
- Hybridoma Production: Spleen cells from the immunized mice are fused with myeloma cells to create hybridoma cells that can produce the desired monoclonal antibody.
- Screening and Selection: Hybridoma cells are screened for the production of antibodies that specifically bind to the Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma. The clone PAT4B9AT is selected for its high specificity and affinity.
- Antibody Purification: The monoclonal antibody is purified from the culture supernatant of the hybridoma cells using techniques such as protein A/G affinity chromatography.
The Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma is involved in various biochemical pathways and interacts with multiple proteins. Some key interactions and reactions include:
- Phosphorylation: The 14-3-3 proteins, including the gamma isoform, bind to phosphorylated serine/threonine residues on target proteins, modulating their activity and stability.
- Signal Transduction: By interacting with various kinases and phosphatases, the 14-3-3 proteins play a pivotal role in signal transduction pathways, influencing cellular responses to external stimuli.
- Apoptosis Regulation: The 14-3-3 proteins can sequester pro-apoptotic factors in the cytoplasm, preventing them from inducing cell death and thus promoting cell survival.
The monoclonal antibody clone PAT4B9AT is widely used in research to study the expression, localization, and function of the Tyr-3/Trp-5 Monooxygenase Activation Protein Gamma in human cells. It is utilized in techniques such as Western blotting, immunoprecipitation, and immunofluorescence to investigate the role of this protein in various cellular processes and disease states.
