YWHAE Antibody

What is YWHAE protein and why is it important in cellular function?

YWHAE (14-3-3 epsilon) functions as an adapter protein implicated in regulating both general and specialized signaling pathways. It binds to numerous partner proteins, typically through recognition of phosphoserine or phosphothreonine motifs, which generally results in modulation of the binding partner's activity . The protein plays crucial roles in:

• Regulating protein HSF1 nuclear export to the cytoplasm

• Participating in antiviral signaling pathways upstream of TBK1 via RIGI interaction

• Directing RIGI redistribution from cytosol to mitochondrial associated membranes

• Inhibiting proliferation and promoting cell cycle arrest by exporting HNRNPC from the nucleus to the cytoplasm for ubiquitin-mediated degradation

In pathological contexts, YWHAE interacts with proteins like DISC1 in schizophrenia, affecting neuronal migration and synaptic function .

What are the common applications for YWHAE antibodies in research?

Based on the available search results, YWHAE antibodies are commonly used in:

• Western blotting (WB) - For protein quantification and detection

• Immunocytochemistry/Immunofluorescence (ICC/IF) - For protein localization in cultured cells

• Immunohistochemistry (IHC-P) - For tissue expression analysis in paraffin-embedded samples

• Flow Cytometry - For protein detection in individual cells

• Immunoprecipitation (IP) - For protein-protein interaction studies

What species reactivity is typically available for YWHAE antibodies?

Most commercially available YWHAE antibodies react with human samples, with many also showing cross-reactivity with mouse and rat samples due to the high conservation of the protein sequence across species . Some antibodies also show predicted reactivity with other species:

The high level of sequence homology (often 100%) across species makes many YWHAE antibodies suitable for cross-species applications .

What is the difference between polyclonal and monoclonal YWHAE antibodies?

The search results show both types are available for YWHAE detection:

Polyclonal YWHAE antibodies:

• Generated from multiple B-cell lineages in immunized animals (often rabbits)

• Recognize multiple epitopes on the YWHAE protein

• Examples include ab137862 (rabbit polyclonal) and AVARP02058_P050 (rabbit polyclonal)

• Suitable for applications where greater sensitivity may be needed

Monoclonal YWHAE antibodies:

• Derived from a single B-cell clone (e.g., from hybridization of mouse FO myeloma cells with spleen cells from immunized BALB/c mice)

• Recognize a single epitope on the YWHAE protein

• Examples include ab92311 (rabbit recombinant monoclonal) and ANT-379 (mouse monoclonal, clone PAT4F8AT)

• Typically offer greater specificity and lower batch-to-batch variation

The choice between polyclonal and monoclonal depends on the specific research application, with monoclonals often preferred for reproducibility in long-term projects.

What are the optimal protocols for immunohistochemistry using YWHAE antibodies?

Based on the methodologies described in the search results, an optimized protocol for immunohistochemistry with YWHAE antibodies includes:

• Sample preparation: Fix histopathological specimens with 10% formalin solution and embed in paraffin

• Sectioning: Cut into 5 μm serial slices

• Deparaffinization: Treat paraffin sections with xylene

• Rehydration: Use gradient alcohol solutions

• Antigen retrieval: Heat samples to unmask antigens

• Blocking: Apply H₂O₂, followed by goat serum blocking solution

• Primary antibody incubation: Add anti-YWHAE antibody (typically at 1:100 dilution, e.g., sc-23957) and incubate overnight at 4°C

• Secondary antibody incubation: Apply HRP-labeled goat anti-rabbit/mouse secondary antibodies

• Detection: Visualize using 3,3-diaminobenzidine (DAB)

• Counterstaining: Use hematoxylin to stain nuclei blue

• Processing: Dehydrate, clear with xylene, and mount

For scoring and evaluation, samples should be independently assessed by two pathologists using a combined scoring system factoring both the proportion of positive cells (0-4 points) and staining intensity (0-3 points) to generate a final score.

How should cellular immunofluorescence be performed with YWHAE antibodies?

The search results provide this optimized protocol for immunofluorescence:

• Cell preparation: Seed cells on microscope slides and allow adherence

• Washing: Wash with phosphate-buffered saline (PBS)

• Blocking: Apply goat serum blocking solution

• Primary antibody incubation: Add anti-YWHAE antibody (typically at 1:100 dilution, e.g., sc-23957)

• Secondary antibody incubation: Apply fluorophore-labeled secondary antibodies (e.g., tetramethylrhodamine-labeled goat anti-rabbit IgG or fluorescein isothiocyanate-labeled goat anti-mouse IgG) and incubate for 2 hours in the dark

• Nuclear staining: Use 4′,6-diamidino-2-phenylindole (DAPI)

• Preparation for imaging: Add anti-quenching agent

• Visualization: View using a confocal microscope

This protocol can be adapted for co-localization studies by including antibodies against interacting proteins in the same preparation.

What are the recommended conditions for YWHAE antibody storage?

Proper storage is crucial for maintaining antibody functionality. Based on the search results, the following storage recommendations apply:

• Short-term storage (up to 1 month): Store at 4°C

• Long-term storage: Store at -20°C, preferably in small aliquots to prevent freeze-thaw cycles

• Formulation: Typically provided in PBS with preservatives such as:

  • PBS with 0.02% sodium azide, 50% glycerol, pH 7.3

  • 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose

The shelf life is approximately 12 months at -20°C and 1 month at 4°C . Repeated freeze-thaw cycles should be avoided as they can degrade antibody performance.

How can YWHAE antibodies be utilized in protein-protein interaction studies?

YWHAE antibodies are valuable tools for investigating protein-protein interactions through immunoprecipitation techniques. Based on the search results, here's an optimized protocol:

• Cell preparation: Collect cells in exponential growth phase and wash

• Lysis: Add ice-cold lysis buffer and sonicate the cell suspension

• Sample preparation: Collect supernatant and determine total protein concentration

• Antibody incubation: Incubate samples (approximately 500 μg) with 2 μg of YWHAE primary antibody (e.g., sc-23957)

• Controls: Include an IgG antibody of the same species as a negative control

• Incubation: Rotate samples slowly overnight at 4°C

• Bead addition: Add Protein A/G Plus-Agarose Beads and incubate for 6 hours

• Collection: Collect bound proteins via centrifugation

• Denaturation: Add loading buffer and boil to denature the proteins

• Analysis: Perform Western blot analysis to identify interacting proteins

This approach has been successfully used to identify YWHAE interactions with various proteins, including RAF1, BRAF, YAP, and TAZ in different cell types, revealing YWHAE's role in multiple signaling pathways .

What is the role of YWHAE in cancer progression and how can antibodies help investigate this?

YWHAE is implicated in cancer progression, particularly in ovarian cancer. Research using YWHAE antibodies has shown:

• YWHAE is substantially upregulated in ovarian cancer tissues

• It represents a risk factor for ovarian cancer prognosis

• YWHAE may influence the malignant behavior of cancer cells

Antibody-based techniques to investigate YWHAE's role in cancer include:

• Expression analysis: Using immunohistochemistry to assess YWHAE expression levels in tumor vs. normal tissues

• Functional studies: Combining antibody detection with YWHAE overexpression or knockdown experiments

• Mechanistic investigations: Using co-immunoprecipitation with YWHAE antibodies to identify cancer-relevant interaction partners

In a specific study, these approaches revealed that YWHAE acts as an HE4 interacting protein that can influence the malignant behavior of ovarian cancer .

How can CRISPR knockout validation be performed using YWHAE antibodies?

YWHAE antibodies are essential for validating CRISPR knockout models. Based on the search results, a methodology for CRISPR KO of YWHAE includes:

• Transfection: Transfect target cells (e.g., NCI-H929 and KMS-11) with 2 μg pspCas9-GFP-YWHAE or pspCas9-GFP control using appropriate nucleofection kit

• Cell sorting: After 2 days, sort GFP+ cells and establish single-cell cultures

• Validation: Confirm knockout using both:

  • Quantitative PCR (to verify gene disruption)

  • Western blot with YWHAE antibodies (to confirm absence of protein expression)

• Control experiments: Include parental cells and cells transfected with empty vector as controls

• Functional assays: Use confirmed KO clones for downstream experiments

For addback experiments to confirm phenotype specificity:

• Transduce WT or YWHAE-KO cells with empty vector or YWHAE-expressing vector

• Select with appropriate antibiotic (e.g., 10 μg/mL blasticidin)

• Validate expression by Western blot using YWHAE antibodies

What is the significance of YWHAE-NUTM2 fusion proteins in cancer and how can antibodies detect these?

YWHAE-NUTM2 fusion proteins play significant roles in oncogenic processes, particularly in endometrial stromal sarcomas. Research using antibodies has revealed:

• YWHAE-NUTM2 oncoprotein regulates proliferation and cyclin D1 expression

• It dysregulates RAF/MEK/MAPK and Hippo/YAP-TAZ signaling pathways

• The fusion protein interacts with RAF1 and BRAF in ESS1 cells

Detection methods using antibodies include:

• Co-immunoprecipitation: Immunoprecipitating RAF1 and BRAF and then immunoblotting for YWHAE to detect the fusion protein (approximately 140/110 kDa isoform)

• Expression systems: Using lentiviral YWHAE-NUTM2-FLAG constructs for studying protein interactions

• Knockdown validation: Using lentiviral shRNA constructs targeting YWHAE-NUTM2 and confirming by immunoblotting

• Interaction studies: Demonstrating YAP/TAZ interactions with YWHAE-NUTM2 (140 kDa band) in various cell lines

Importantly, research has shown that YAP/TAZ interactions with YWHAE-NUTM2 appear stronger than those reported previously for YWHAE alone.

What are common causes of non-specific binding when using YWHAE antibodies?

Non-specific binding can compromise experimental results. Based on the search results and general antibody principles, common causes include:

• Insufficient blocking: Inadequate blocking can lead to high background. Ensure proper blocking with appropriate serum (e.g., goat serum as used in reported protocols)

• Antibody concentration: Too high antibody concentration increases non-specific binding. Titrate antibodies to find optimal dilution (typical working dilutions range from 1:100 to 1:1500 for YWHAE antibodies)

• Cross-reactivity: YWHAE belongs to the 14-3-3 family with seven highly conserved isoforms (β, γ, ε, σ, ζ, τ, and η) . Choose antibodies raised against unique epitopes to minimize cross-reactivity

• Sample preparation issues: Improper fixation or antigen retrieval can lead to artifactual staining

• Secondary antibody problems: Ensure secondary antibodies match the host species of the primary antibody

To address these issues, always include appropriate controls:

• Negative controls using isotype-matched IgG (e.g., 5145S, Cell Signaling Technology)

• Peptide competition assays using blocking peptides (e.g., AAP30642 for AVARP02058_P050)

How can researchers validate the specificity of YWHAE antibodies?

Validation is crucial for ensuring experimental reliability. Methods include:

• Western blot analysis: Confirm a single band of expected molecular weight (29-32 kDa for YWHAE)

• CRISPR knockout controls: Generate YWHAE knockout cells as negative controls (as described in the multiple myeloma study)

• siRNA knockdown: Transfect cells with YWHAE-siRNA (e.g., 5′-GAAGCAGGUUAGCGUUGAATT-3′) and confirm reduced signal compared to mock-siRNA controls

• Overexpression models: Compare cells with endogenous YWHAE levels to those overexpressing the protein

• Peptide competition: Pre-incubate antibody with immunizing peptide before application to samples; this should abolish specific staining

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of YWHAE and verify consistent results

These validation steps are particularly important when studying YWHAE in cancer contexts, where expression levels may be altered or when investigating fusion proteins like YWHAE-NUTM2.

What strategies can help optimize immunoprecipitation experiments with YWHAE antibodies?

Immunoprecipitation is a powerful technique for studying YWHAE interactions but requires optimization:

• Antibody selection: Choose antibodies validated for immunoprecipitation applications

• Lysis conditions: Optimize buffer composition to preserve protein-protein interactions while efficiently extracting YWHAE

• Pre-clearing: Remove non-specific binding proteins by pre-incubating lysates with beads before adding antibody

• Antibody amount optimization: Titrate antibody amounts (typically 2 μg is used for YWHAE IP)

• Controls: Always include:

  • IgG control from the same species as the primary antibody

  • Input sample (typically 5-10% of the amount used for IP)

  • When possible, a YWHAE knockout or knockdown sample

This approach has successfully identified novel YWHAE interactions in cancer research, revealing its role in ovarian cancer malignancy through interaction with HE4 protein and the function of YWHAE-NUTM2 fusion proteins through interactions with RAF1, BRAF, YAP, and TAZ .

What scoring systems are recommended for YWHAE immunohistochemistry in clinical samples?

For clinical and translational research, standardized scoring of YWHAE expression is crucial. Based on search results, a recommended scoring system includes:

• Positive cell proportion score:

  • Less than 5% positive cells: 0 points

  • 5-25% positive cells: 1 point

  • 26-50% positive cells: 2 points

  • 51-75% positive cells: 3 points

  • More than 75% positive cells: 4 points

• Staining intensity score:

  • No staining: 0 points

  • Light yellow: 1 point

  • Brownish-yellow: 2 points

  • Brown: 3 points

• Final score calculation: Multiply proportion score by intensity score (range: 0-12)

• Interpretation:

  • 0-2 points: Negative expression (−)

  • 3-4 points: Weakly positive expression (+)

  • 5-8 points: Moderately positive expression (++)

  • 9-12 points: Strongly positive expression (+++)

For clinical relevance, it's recommended that samples be independently scored by two pathologists blinded to clinical information, with a third pathologist resolving discordant results.

What control samples are recommended for YWHAE antibody experiments?

For rigorous research with YWHAE antibodies, appropriate controls include:

• Positive controls: Tissues or cell lines known to express YWHAE (the protein is widely expressed but with tissue-specific levels)

• Negative controls:

  • YWHAE knockout cell lines generated via CRISPR (as described in the multiple myeloma study)

  • YWHAE knockdown cells using validated siRNA constructs (e.g., 5′-GAAGCAGGUUAGCGUUGAATT-3′)

  • Isotype-matched IgG antibodies for immunoprecipitation experiments (e.g., 5145S, Cell Signaling Technology)

• Technical controls:

  • Secondary antibody-only controls to assess background

  • For immunofluorescence, include single-stained samples when performing co-localization studies

• Validation controls:

  • Cells transfected with YWHAE-overexpression constructs

  • Western blot analysis confirming antibody specificity by showing a band of expected molecular weight (29-32 kDa)