YWHAG Antibody

What is YWHAG and what are its known functions in cellular processes?

YWHAG is a member of the 14-3-3 protein family in mammals that regulates diverse cellular processes including cell cycle progression, protein transport, cell survival, and apoptosis . As an adaptor protein, YWHAG acts as a molecular scaffold that facilitates protein-protein interactions by binding to phosphorylated serine/threonine motifs. In normal cellular physiology, YWHAG plays crucial roles in signal transduction pathways by interacting with numerous partner proteins to modulate their activity, subcellular localization, or stability. Its function is particularly important in maintaining cellular homeostasis through regulation of metabolic processes and stress responses .

What are the standard applications for YWHAG antibodies in research?

YWHAG antibodies are primarily utilized in the following research applications:

• Western blot (WB): For detecting and quantifying YWHAG protein expression in cell or tissue lysates

• Immunohistochemistry (IHC): For visualizing YWHAG expression patterns in tissue sections

• Immunofluorescence: For subcellular localization studies, often used in co-localization experiments with other proteins

When selecting a YWHAG antibody, researchers should consider its validated applications. For instance, rabbit polyclonal antibodies to YWHAG have demonstrated efficacy in both Western blot and immunohistochemistry applications across human, mouse, and rat samples .

How should researchers store and handle YWHAG antibodies for optimal results?

YWHAG antibodies require specific storage and handling conditions to maintain their efficacy:

• Storage temperature: Store at -20°C for long-term preservation

• Formulation: Typically supplied at concentrations around 1.0 mg/mL in phosphate-buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody upon first thaw

• Working dilutions: Prepare fresh working dilutions on the day of the experiment

Proper adherence to these storage and handling guidelines ensures antibody stability and consistent experimental results across multiple studies.

What are the optimal protocols for using YWHAG antibodies in Western blot applications?

For Western blot applications using YWHAG antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Extract total protein from cells or tissues using standard lysis buffers

• Protein separation: Use SDS-PAGE with 10-12% acrylamide gels for optimal separation

• Transfer: Transfer proteins to PVDF membranes using standard wet or semi-dry transfer systems

• Blocking: Block membranes with 5% skim milk for 2 hours at room temperature

• Primary antibody incubation: Dilute YWHAG antibody (typical working dilution 1:500) and incubate overnight at 4°C

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG) for 1 hour at room temperature

• Detection: Visualize using enhanced chemiluminescence systems

Validation experiments show that rabbit polyclonal YWHAG antibodies effectively detect endogenous levels of total YWHAG protein in various cell lines .

How can researchers optimize immunohistochemistry protocols for YWHAG detection?

For effective immunohistochemical detection of YWHAG in tissue sections:

• Tissue preparation: Fix tissues in 4% paraformaldehyde and embed in paraffin

• Sectioning: Cut 4-6 μm thick sections and mount on positively charged slides

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ and non-specific binding with 5% BSA

• Primary antibody: Apply YWHAG antibody at an optimized dilution (typically 1:200) and incubate overnight at 4°C

• Detection system: Use appropriate secondary antibody and visualization system (DAB or fluorescent labels)

• Counterstaining: Counterstain nuclei with hematoxylin for brightfield or DAPI for fluorescence

Published validation data demonstrates successful YWHAG detection in human lung cancer tissue using this approach .

How does YWHAG expression correlate with cancer progression and prognosis?

YWHAG has been identified as a key oncogenic gene in multiple cancer types, with significant implications for disease progression and patient outcomes:

The prognostic value of YWHAG expression has been demonstrated through analysis of The Cancer Genome Atlas (TCGA) database, revealing that high YWHAG expression correlates with poorer outcomes in cervical cancer patients . In experimental models, silencing YWHAG has been shown to diminish primary tumor volumes, prevent metastasis, and prolong median survival periods in mouse models .

What methodological approaches can be used to study YWHAG's role in oxidative stress responses?

To investigate YWHAG's involvement in cellular oxidative stress responses, researchers can employ these methodological approaches:

• ROS measurement assays:

  • Use fluorescent probes (e.g., DCFDA) to measure intracellular ROS levels

  • Compare ROS accumulation in YWHAG-expressing versus YWHAG-deficient cells

• YWHAG knockdown/knockout models:

  • Generate stable YWHAG-silenced cell lines using siRNA or CRISPR/Cas9 technologies

  • Verify knockdown efficiency through qPCR and Western blot

• Autophagy flux assessment:

  • Monitor LC3-I to LC3-II conversion by Western blot

  • Measure autophagic vesicle formation using fluorescence microscopy

  • Assess YWHAG's role in autophagy as a mechanism protecting against oxidative catastrophe

• Cell viability and death assays:

  • Measure apoptosis markers in response to oxidative stress with and without YWHAG expression

  • Assess cell proliferation using assays such as CCK-8

Research has demonstrated that YWHAG deficiency results in rapid accumulation of reactive oxygen species (ROS), delayed epithelial-mesenchymal transition (EMT), and increased cell death . This suggests YWHAG plays a cytoprotective role against oxidative stress during cancer progression.

How does YWHAG interact with metabolic pathways in cancer cells?

YWHAG has been implicated in modulating cancer cell metabolism, particularly through its interaction with the pentose phosphate pathway (PPP):

• YWHAG influences glucose metabolism:

  • Knockout of YWHAG reduces glucose uptake in cancer cells

  • Affects metabolites of the pentose phosphate pathway, including D-sedoheptulose-1,7-biphosphate, deoxyribose-phosphate, fructose 1,6-biphosphate, and ribose-5-phosphate

• Investigation methods:

  • Metabolite profiling using mass spectrometry

  • Glucose uptake assays using fluorescent glucose analogs

  • Expression analysis of rate-limiting enzymes in the PPP

• Mechanistic pathways:

  • YWHAG positively correlates with HIF-1α expression in cervical cancer samples

  • Co-expression and interaction with HIF-1α in the cytoplasm has been demonstrated through immunofluorescence assays

Experimental evidence shows that after YWHAG knockout, the expression levels of PPP-related proteins decrease, and glucose uptake is reduced . This suggests YWHAG plays a critical role in cancer cell metabolism by promoting the pentose phosphate pathway, which is essential for nucleotide synthesis and NADPH production for managing oxidative stress.

How can researchers validate YWHAG antibody specificity for their experimental models?

Ensuring antibody specificity is crucial for generating reliable research data. For YWHAG antibodies, researchers should implement these validation approaches:

• Positive and negative controls:

  • Use cell lines or tissues with known YWHAG expression levels

  • Include YWHAG-knockout or knockdown samples as negative controls

• Western blot validation:

  • Confirm detection of a single band at the expected molecular weight (~28 kDa)

  • Perform peptide competition assays with the immunizing peptide

• Cross-reactivity assessment:

  • Test the antibody against other 14-3-3 family members to ensure specificity

  • Verify reactivity across relevant species (human, mouse, rat) if conducting cross-species research

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that immunoprecipitated proteins are indeed YWHAG through peptide sequencing

Published validation data for commercial YWHAG antibodies typically include Western blot analysis showing specific detection in various cell lines and immunohistochemistry demonstrating appropriate tissue localization patterns .

What are common challenges when using YWHAG antibodies and how can they be addressed?

Researchers commonly encounter these challenges when working with YWHAG antibodies:

• Cross-reactivity with other 14-3-3 isoforms:

  • Solution: Use antibodies raised against unique peptide sequences of YWHAG

  • Validate specificity through knockout/knockdown controls

• Background signal in immunostaining:

  • Solution: Optimize blocking conditions (use 5% BSA instead of serum)

  • Increase washing steps and durations

  • Test different antibody dilutions

• Variable expression levels across tissues:

  • Solution: Adjust exposure times for Western blot detection

  • Optimize antibody concentration for different tissue types

• Epitope masking due to protein-protein interactions:

  • Solution: Test different sample preparation methods that may disrupt protein complexes

  • Consider native versus denaturing conditions depending on the research question

• Reproducibility issues:

  • Solution: Use antibodies from consistent lots

  • Standardize protocols across experiments

  • Document detailed methods for improved reproducibility

How is YWHAG being investigated as a therapeutic target in cancer?

Recent research has highlighted YWHAG as a potential therapeutic target in cancer treatment strategies:

• Small molecule inhibitors:

  • Compounds like curcumenol have been investigated for targeting YWHAG

  • Curcumenol can inhibit YWHAG and increase sensitivity to cisplatin chemotherapy in cervical cancer

• Combinatorial approaches:

  • YWHAG inhibition combined with conventional chemotherapy shows enhanced efficacy

  • Targeting YWHAG increases cisplatin-induced apoptosis in cancer cells

• EMT and metastasis prevention:

  • Silencing YWHAG diminishes primary tumor volumes and prevents metastasis in animal models

  • YWHAG is integral to initiate and maintain cancer epithelial-mesenchymal transition

• Autophagy modulation:

  • YWHAG protects cancer cells from oxidative catastrophe during EMT by increasing autophagic flux

  • Disruption of the YWHAG regulome abolishes EMT, effectively limiting secondary tumor growth

The emerging understanding of YWHAG's role in protecting cancer cells during EMT through enhanced autophagy suggests that deliberate disruption of YWHAG regulome could be a promising novel strategy to curb tumor metastasis .

What are the latest techniques for studying YWHAG's protein-protein interactions in complex biological systems?

Cutting-edge methodologies for investigating YWHAG's interactome include:

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal interacting partners of YWHAG in living cells

  • TurboID for rapid labeling of protein interaction networks

• Advanced microscopy approaches:

  • Super-resolution microscopy for visualizing YWHAG interactions at nanoscale resolution

  • Förster resonance energy transfer (FRET) to detect direct protein-protein interactions

  • Fluorescence correlation spectroscopy to analyze binding dynamics

• Proteomics and bioinformatics:

  • Affinity purification coupled with mass spectrometry

  • Construction of YWHAG regulomes from kinomic, transcriptomic, and interactome data

  • Network analysis to identify hub genes and critical pathways

• Functional screening approaches:

  • CRISPR screens to identify synthetic lethal interactions with YWHAG

  • Phosphoproteomic analysis to identify YWHAG-dependent phosphorylation events

Recent research has employed these advanced techniques to reveal that YWHAG interacts with HIF-1α in the cytoplasm, with positive correlation demonstrated through both bioinformatic analysis of TCGA data and experimental validation using immunofluorescence co-staining assays .