esrp2 Antibody

What is ESRP2 and why is it important to study in research?

ESRP2 functions as an mRNA splicing factor that regulates the formation of epithelial cell-specific isoforms. It specifically regulates the expression of FGFR2-IIIb, an epithelial cell-specific isoform of FGFR2, and controls the splicing of CD44, CTNND1, and ENAH transcripts that undergo changes during the epithelial-to-mesenchymal transition (EMT) . Studying ESRP2 is crucial for understanding splicing regulation mechanisms in epithelial biology and their dysregulation in pathological conditions like cancer, where alternative splicing patterns significantly influence disease progression .

What are the key characteristics of the ESRP2 protein?

The human ESRP2 protein has the following characteristics:

• Canonical protein length: 727 amino acid residues

• Molecular weight: 78.4 kDa (observed in Western blots)

• Subcellular localization: Nucleus

• Number of reported isoforms: Up to 2

• Expression pattern: Epithelial cell-specific

• Function: Binds GU-rich sequence motifs in the ISE/ISS-3 cis-element regulatory region in mRNAs

• Protein family: ESRP family

• Alternative names: RNA-binding motif protein 35B (RBM35B), RNA-binding protein 35B

What applications are ESRP2 antibodies commonly used for?

ESRP2 antibodies are validated for multiple laboratory applications:

These applications enable researchers to detect, localize, and quantify ESRP2 expression in various experimental contexts .

Which species reactivity should be considered when selecting an ESRP2 antibody?

While many ESRP2 antibodies are primarily validated for human samples, cross-reactivity varies significantly between products:

Researchers should carefully verify the specific cross-reactivity claims and validation data for their species of interest, especially when working with non-human models .

How should researchers optimize Western blot protocols for ESRP2 detection?

For optimal Western blot detection of ESRP2:

• Sample preparation: Use whole cell lysates from epithelial cells (A431, HeLa, HepG2 have shown good endogenous expression)

• Protein loading: Load 30 μg of total protein per lane

• Gel percentage: Use 7.5% SDS-PAGE for optimal separation around the 78-79 kDa range

• Transfer conditions: Transfer to PVDF or nitrocellulose membranes using standard protocols

• Blocking: Block with 5% non-fat milk or BSA in TBST

• Primary antibody: Dilute antibody according to manufacturer's recommendation (typically 1:500-1:1000); incubate overnight at 4°C

• Secondary antibody: Use HRP-conjugated anti-rabbit or anti-mouse IgG (depending on primary antibody host)

• Detection: Use ECL-based detection systems

• Controls: Include positive control lysates (epithelial cell lines) and negative controls (mesenchymal cell lines with low ESRP2 expression)

• Expected band size: Look for bands at approximately 78-79 kDa

What are the critical parameters for immunohistochemical detection of ESRP2?

For successful IHC detection of ESRP2 in tissue samples:

• Tissue fixation: Standard formalin-fixed, paraffin-embedded (FFPE) tissues are suitable

• Sectioning: 4-5 μm sections recommended

• Antigen retrieval: Critical step; use high-pressure retrieval in citrate buffer (pH 6.0) or TE buffer (pH 9.0)

• Blocking: Block with 10% normal serum (goat or horse depending on secondary antibody) for 30 minutes at room temperature

• Primary antibody: Dilute 1:200-1:300; incubate overnight at 4°C in 1% BSA solution

• Detection system: Biotin-streptavidin or polymer-based detection systems work well

• Visualization: DAB substrate for brown coloration

• Counterstaining: Hematoxylin for nuclear visualization

• Positive controls: Breast cancer tissue or adrenal gland show reliable ESRP2 expression

• Expected pattern: Nuclear staining in epithelial cells

How can researchers validate the specificity of their ESRP2 antibody?

To ensure ESRP2 antibody specificity:

• Multiple antibody approach: Use at least two antibodies targeting different epitopes of ESRP2

• Knockdown/knockout validation: Compare staining in ESRP2 knockdown or knockout cells versus wild-type

• Overexpression validation: Check for increased signal in ESRP2-overexpressing cells

• Cross-reactivity testing: Test against related proteins, especially ESRP1, to ensure specificity

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Western blot correlation: Confirm that IHC/IF results correlate with Western blot data

• Size verification: Confirm the detected protein is the expected molecular weight (78-79 kDa)

• Cell/tissue distribution: Verify that staining matches known epithelial-specific expression pattern

• Isoform specificity: Determine if the antibody detects all ESRP2 isoforms or is isoform-specific

• Batch-to-batch consistency: Test new antibody lots against previous validated lots

How can ESRP2 antibodies be used to study epithelial-to-mesenchymal transition?

ESRP2 antibodies are valuable tools for investigating EMT processes:

• Temporal expression analysis: Track ESRP2 downregulation during EMT induction using Western blot or immunofluorescence

• Spatial expression patterns: Use IHC to examine ESRP2 expression at tumor invasive fronts versus tumor centers

• Co-localization studies: Combine ESRP2 antibodies with E-cadherin and other EMT markers in multiplexed immunofluorescence

• Reversibility assessment: Monitor ESRP2 re-expression during mesenchymal-to-epithelial transition (MET)

• Metastasis research: Compare ESRP2 expression in primary tumors versus metastatic sites

• Mechanistic studies: Use ESRP2 antibodies in RNA immunoprecipitation to identify target transcripts

• Therapeutic response: Assess changes in ESRP2 expression following EMT-targeting treatments

• Prognostic evaluation: Correlate ESRP2 expression with patient outcomes in clinical samples

Research has shown that ESRP2 expression is plastic during carcinogenesis—upregulated in oral squamous cell carcinoma compared to normal epithelium, downregulated at invasive fronts, and re-expressed in lymph node metastases, making it a valuable marker for tracking EMT-MET dynamics in cancer progression .

What approaches can resolve contradictory ESRP2 expression data in cancer studies?

When facing contradictory ESRP2 expression data:

• Tumor heterogeneity analysis: Use spatial transcriptomics or single-cell analysis alongside IHC to assess intratumoral heterogeneity

• Isoform-specific detection: Employ antibodies targeting specific ESRP2 isoforms if contradictions might be isoform-related

• Context-dependent expression: Examine ESRP2 expression relative to tumor microenvironment factors

• Temporal dynamics: Consider time-course experiments to capture dynamic expression changes

• Technical validation: Compare results using multiple antibodies targeting different epitopes

• Platform comparison: Validate protein expression data with mRNA analysis methods

• Sample preparation effects: Standardize fixation and processing protocols to minimize technical variability

• Quantitative analysis: Use digital pathology for objective quantification rather than subjective scoring

• Cell type specificity: Use cell type markers to ensure comparing equivalent cell populations

• Alternative splicing feedback: Consider that ESRP2 itself might be alternatively spliced in different contexts

This multi-faceted approach is especially important given reports of context-dependent ESRP2 expression in various cancers, including pancreatic ductal adenocarcinoma, oral squamous carcinoma, ovarian cancer, and luminal-type breast cancer .

How can ESRP2 antibodies be utilized in RNA splicing research?

For investigating ESRP2's role in RNA splicing:

• RNA-immunoprecipitation (RIP): Use ESRP2 antibodies to pull down ESRP2-bound RNA targets

• Cross-linking immunoprecipitation (CLIP): Identify direct RNA binding sites using UV cross-linking followed by ESRP2 immunoprecipitation

• Alternative splicing analysis: Couple ESRP2 knockdown with splicing-sensitive RT-PCR or RNA-seq to identify ESRP2-dependent splicing events

• Co-immunoprecipitation: Identify ESRP2 protein interactors in the splicing machinery

• Subcellular fractionation: Track ESRP2 localization in nuclear splicing compartments

• Splicing reporter assays: Use minigene constructs to measure ESRP2's effect on specific splicing events

• Mass spectrometry: Identify post-translational modifications of ESRP2 that might regulate its splicing activity

• Chromatin immunoprecipitation: Investigate if ESRP2 has co-transcriptional splicing roles

• Live cell imaging: Use fluorescently tagged antibodies to track ESRP2 dynamics during splicing

• Proximity ligation assays: Detect interactions between ESRP2 and core splicing factors in situ

These approaches can help elucidate how ESRP2 specifically regulates alternative splicing of key transcripts like FGFR2, CD44, CTNND1, and ENAH that undergo changes during EMT .

What methodological considerations are important when comparing ESRP1 and ESRP2 functions?

To effectively compare ESRP1 and ESRP2 functions:

• Antibody specificity: Use antibodies validated for specificity between these paralogous proteins

• Expression correlation: Analyze if ESRP1 and ESRP2 are co-expressed or differentially expressed across tissues

• Knockout models: Compare single knockouts versus double knockouts to assess functional redundancy

• Rescue experiments: Test if ESRP1 can rescue ESRP2 knockout phenotypes and vice versa

• Domain-specific function: Use antibodies targeting specific functional domains to understand structural contributions

• Cross-regulatory effects: Examine if depleting one ESRP affects expression of the other

• Splicing target comparison: Identify shared versus unique RNA targets using CLIP-seq with specific antibodies

• Binding affinity analyses: Compare RNA binding affinities using purified proteins and target sequences

• Post-translational regulation: Investigate if ESRP1 and ESRP2 are differentially regulated by PTMs

• Evolutionary conservation: Compare conservation of binding motifs and functional domains between species

Research has shown that while ESRP1 and ESRP2 have overlapping functions, they may also regulate cell motility through distinct transcriptional and/or post-transcriptional mechanisms, highlighting the importance of studying both proteins individually and in combination .

What are common challenges in Western blot detection of ESRP2 and how can they be addressed?

Common Western blot issues and solutions:

• No signal:

  • Verify ESRP2 expression in your cell line (epithelial cell lines like A431, HeLa, HepG2 are good positive controls)

  • Increase antibody concentration or extend incubation time

  • Ensure sample preparation preserves nuclear proteins (where ESRP2 is localized)

  • Check transfer efficiency for high molecular weight proteins

• Multiple bands:

  • Determine if bands represent different isoforms (ESRP2 has at least 2 reported isoforms)

  • Optimize gel percentage for better resolution around 78-79 kDa

  • Increase washing steps to reduce non-specific binding

  • Use freshly prepared lysates to minimize degradation products

• High background:

  • Increase blocking time/concentration

  • Dilute primary antibody further

  • Use highly purified antibody preparations

  • Add 0.1-0.5% Tween-20 to washing and antibody diluent buffers

How can researchers interpret variable ESRP2 staining patterns in immunohistochemistry?

Interpreting variable IHC staining patterns:

• Heterogeneous nuclear staining:

  • May reflect biological heterogeneity in ESRP2 expression

  • Correlate with epithelial differentiation markers

  • Consider quantifying percentage of positive cells in different regions

• Variable staining intensity:

  • Standardize fixation time to minimize technical variability

  • Optimize antigen retrieval conditions (compare citrate buffer pH 6.0 vs. TE buffer pH 9.0)

  • Consider quantitative image analysis for objective intensity measurement

• Unexpected cytoplasmic staining:

  • Verify antibody specificity with additional validation methods

  • Consider potential shuttling between nucleus and cytoplasm in certain contexts

  • Compare with mRNA localization by RNA in situ hybridization

What strategies can improve reproducibility in ESRP2 immunofluorescence experiments?

For improved reproducibility in immunofluorescence:

• Fixation optimization:

  • Compare paraformaldehyde, methanol, and acetone fixation for optimal epitope preservation

  • Standardize fixation time and temperature

• Signal amplification:

  • Consider tyramide signal amplification for low abundance detection

  • Use biotin-streptavidin systems for enhanced sensitivity

• Autofluorescence reduction:

  • Include Sudan Black B treatment to reduce autofluorescence

  • Optimize imaging settings to distinguish true signal from background

• Multiplexed detection:

  • Use antibodies raised in different host species for co-localization studies

  • Include appropriate controls for spectral overlap

• Quantitative analysis:

  • Implement automated image analysis algorithms for objective quantification

  • Establish clear intensity thresholds for positive vs. negative staining

How can ESRP2 antibodies be utilized in cancer biomarker research?

Applications of ESRP2 antibodies in cancer biomarker research:

• Prognostic stratification: Correlate ESRP2 expression levels with patient outcomes in different cancer types

• Predictive biomarker development: Investigate if ESRP2 expression correlates with response to specific therapies

• Multi-marker panels: Combine ESRP2 with other EMT markers for improved prognostic accuracy

• Liquid biopsy development: Explore if circulating tumor cells with high ESRP2 expression have different metastatic potential

• Spatial heterogeneity mapping: Use multiplexed IHC to map ESRP2 expression across tumor regions

• Therapy response monitoring: Track changes in ESRP2 expression during treatment

• Cancer subtyping: Determine if ESRP2 expression helps define molecular subtypes with distinct behaviors

• Metastasis prediction: Compare ESRP2 expression in primary tumors that do or do not metastasize

Research has documented overexpression of ESRP2 in various malignant tumors, including pancreatic ductal adenocarcinoma, oral squamous carcinoma, ovarian cancer, and luminal-type breast cancer, highlighting its potential value as a biomarker .

What novel techniques are enhancing the utility of ESRP2 antibodies in splicing research?

Emerging techniques improving ESRP2 antibody applications:

• Single-molecule imaging: Track individual ESRP2-RNA interactions in real-time using fluorescently labeled antibodies

• Super-resolution microscopy: Visualize ESRP2 distribution within nuclear speckles at nanometer resolution

• In situ proximity ligation: Detect dynamic interactions between ESRP2 and splicing machinery components

• Mass cytometry (CyTOF): Multiplex ESRP2 with dozens of other markers for high-dimensional single-cell analysis

• Spatial transcriptomics: Correlate ESRP2 protein localization with local splicing outcomes

• Targeted proteomics: Develop quantitative mass spectrometry assays using antibody-based enrichment

• CRISPR screens: Combine with ESRP2 antibodies to identify functional regulators of ESRP2

• Organoid models: Track ESRP2 expression during 3D epithelial differentiation

• Patient-derived xenografts: Study ESRP2 expression in models preserving tumor heterogeneity

• Digital spatial profiling: Quantify ESRP2 with spatial context in the tumor microenvironment

These advanced techniques are expanding our understanding of ESRP2's dynamic role in regulating alternative splicing in both normal epithelial biology and disease states.

How do post-translational modifications affect ESRP2 detection by antibodies?

Post-translational modifications (PTMs) can significantly impact ESRP2 antibody detection:

• Epitope masking: PTMs may directly block antibody binding sites

• Conformational changes: PTMs can alter protein folding, affecting accessibility of distant epitopes

• Fragmentation effects: Some PTMs might trigger proteolytic processing, resulting in altered band patterns

• Isoform-specific modification: Different ESRP2 isoforms may have distinct PTM patterns

• Context-dependent modifications: Stress, cell cycle phase, or disease state may induce specific PTMs

• Modification-specific antibodies: Consider developing antibodies specifically detecting phosphorylated, ubiquitinated, or otherwise modified ESRP2

• Validation approaches: Use phosphatase treatment or other enzymatic removal of PTMs to confirm their impact on detection

• Bioinformatic prediction: Utilize PTM prediction tools to identify potential sites affecting antibody binding

• Mass spectrometry verification: Confirm presence and location of PTMs in your experimental system

• Sample preparation considerations: Preservation of labile PTMs may require specific buffer components (phosphatase inhibitors, deubiquitinase inhibitors)

Understanding these factors is essential for accurate interpretation of ESRP2 detection results, especially when comparing different physiological or pathological states.

How might ESRP2 antibodies contribute to therapeutic development targeting alternative splicing?

Potential contributions to therapeutic development:

• Target validation: Confirm ESRP2 expression in disease contexts amenable to splicing modulation

• Companion diagnostics: Develop IHC-based assays to identify patients likely to respond to splicing-targeted therapies

• Mechanism of action studies: Track changes in ESRP2 localization or expression following treatment with splicing modulators

• Resistance mechanism identification: Determine if altered ESRP2 expression correlates with resistance to therapy

• Combination therapy rationale: Identify optimal drug combinations based on ESRP2 expression patterns

• Delivery system development: Use antibodies to target therapeutic payloads to ESRP2-expressing cells

• Pharmacodynamic markers: Monitor ESRP2-regulated splicing events as indicators of on-target drug activity

• Target engagement studies: Develop assays to confirm binding of compounds to ESRP2

• Off-target effect assessment: Examine if splicing modulators affect related splicing factors beyond ESRP2

• Therapeutic window determination: Compare ESRP2 levels in diseased vs. normal tissues to assess potential toxicity

These applications highlight the translational potential of ESRP2 research beyond basic mechanistic studies.

What are promising directions for developing next-generation ESRP2 detection tools?

Future directions for improved ESRP2 detection:

• Isoform-specific antibodies: Develop tools specifically targeting individual ESRP2 isoforms

• Modification-state antibodies: Create antibodies recognizing specific phosphorylation, ubiquitination or other PTM states

• Intrabodies: Engineer antibody fragments for live-cell tracking of ESRP2

• Nanobodies: Develop smaller binding molecules for improved tissue penetration and resolution

• Aptamer-based sensors: Create nucleic acid aptamers as alternatives to protein-based antibodies

• FRET-based biosensors: Design tools to monitor ESRP2 conformational changes during RNA binding

• Multiplex imaging panels: Develop antibody panels for simultaneous detection of ESRP2 with its RNA targets and interacting proteins

• Antibody-oligonucleotide conjugates: Combine protein detection with RNA sequence identification

• CRISPR knock-in tags: Generate endogenously tagged ESRP2 to overcome antibody limitations

• Mass cytometry reagents: Develop metal-conjugated antibodies for high-dimensional single-cell analysis