ERF14 Antibody

What is ERF14 antibody and what protein does it target?

ERF14 antibody targets the ERF (ETS2 repressor factor) protein, which belongs to the ETS family of transcription factors. The antibody specifically recognizes the ERF protein that functions as a transcriptional repressor in various cellular processes. ERF proteins are involved in cell proliferation, differentiation, and oncogenic transformation . The molecular weight of the targeted protein is approximately 37.3 kilodaltons, and it may also be known by alternative names depending on species and specific variants .

What applications are ERF14 antibodies most commonly used for in research?

ERF14 antibodies are primarily used in several key research applications:

• Western blotting (WB): For detection and quantification of ERF protein in cell or tissue lysates

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

• Immunocytochemistry (ICC) and Immunofluorescence (IF): For cellular localization studies

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

• ELISA: For quantitative detection of ERF in solution

The selection of application depends on the specific research question, with Western blotting and immunohistochemistry being the most frequently utilized techniques for initial characterization studies.

What are the key considerations for antibody validation in ERF14 research?

When validating ERF14 antibodies for research, consider:

• Specificity testing: Confirm target specificity using positive and negative controls including:

  • Knockout/knockdown cell lines

  • Overexpression systems

  • Known positive/negative tissue samples

• Cross-reactivity assessment: Test against related proteins, particularly other ETS family members

• Application-specific validation: Validate for each intended application (WB, IHC, IF) as performance can vary significantly

• Lot-to-lot consistency: Check new lots against reference samples to ensure consistent performance

• Species reactivity: Confirm reactivity with your model organism as antibodies may show differential cross-reactivity across species (human, mouse, rat, etc.)

How do monoclonal and polyclonal ERF14 antibodies differ in research applications?

The choice between monoclonal and polyclonal ERF14 antibodies significantly impacts experimental outcomes:

For studies requiring precise quantification or epitope-specific detection, monoclonal antibodies are preferred. For detection of low-abundance targets or when protein conformation may be altered, polyclonal antibodies often provide advantages .

What are the critical factors affecting ERF14 antibody performance in chromatin immunoprecipitation (ChIP) experiments?

Successful ChIP experiments with ERF14 antibodies depend on several critical factors:

• Antibody quality: ChIP-grade antibodies specifically validated for this application are essential

• Epitope accessibility: The ERF14 epitope must be accessible in the chromatin context

• Crosslinking optimization: Excessive crosslinking can mask epitopes; insufficient crosslinking leads to poor recovery

• Sonication parameters: Must be optimized to generate appropriate DNA fragment sizes (200-600bp)

• Antibody concentration: Titration experiments are necessary to determine optimal antibody:chromatin ratios

• Negative controls: IgG controls and ideally biological controls (knockdown/knockout) should be included

• Washing stringency: Buffer composition affects specificity vs. sensitivity tradeoffs

When troubleshooting ChIP experiments, systematic evaluation of each parameter is recommended, starting with antibody validation using known targets of ERF transcriptional regulation.

How can researchers address epitope masking issues when using ERF14 antibodies?

Epitope masking is a common challenge with ERF14 antibodies, particularly when:

• Post-translational modifications alter the epitope region

• Protein-protein interactions block antibody access

• Conformational changes hide the epitope

• Fixation procedures modify the epitope structure

To address epitope masking:

• Epitope retrieval methods: For FFPE samples, optimize antigen retrieval using:

  • Heat-induced epitope retrieval (HIER) with citrate or EDTA buffers

  • Enzymatic retrieval with proteinase K or trypsin

• Denaturation optimization: For Western blotting:

  • Test different reducing agent concentrations

  • Vary sample heating time and temperature

  • Consider native vs. denaturing conditions

• Alternative antibodies: Use antibodies recognizing different epitopes

• Sample preparation modifications:

  • Adjust fixation times

  • Test different lysis buffers

  • Consider native vs. crosslinked conditions

What are the best practices for optimizing ERF14 antibody dilutions across different applications?

Optimal ERF14 antibody dilution varies significantly by application, antibody type, and sample characteristics. Follow these methodological approaches:

• Systematic titration:

  • Start with manufacturer's recommended range

  • Test 3-4 dilutions in a 2-fold or 5-fold series

  • Expand range based on initial results

• Application-specific starting points:

  Application	Typical Dilution Range
  Western blot	1:500 to 1:5000
  IHC-Paraffin	1:50 to 1:500
  ICC/IF	1:100 to 1:1000
  ELISA	1:1000 to 1:10,000
  Flow cytometry	1:50 to 1:200

• Optimization criteria:

  • For Western blots: Signal-to-noise ratio, specific band with minimal background

  • For IHC/ICC/IF: Clear signal localization with minimal background

  • For IP: Maximum target precipitation with minimal non-specific binding

• Sample-specific adjustments:

  • Increase antibody concentration for low-abundance targets

  • Decrease concentration for highly expressed targets

  • Adjust blocking conditions along with antibody dilution

How should researchers troubleshoot non-specific binding with ERF14 antibodies?

Non-specific binding is a common challenge with ERF14 antibodies. Address this methodically:

• Blocking optimization:

  • Test alternative blocking agents (BSA, milk, serum, commercial blockers)

  • Increase blocking time or concentration

  • Use casein-based blockers for phospho-specific antibodies

• Washing protocol refinement:

  • Increase wash duration or number of washes

  • Add detergents (0.05-0.1% Tween-20 or Triton X-100)

  • Test high-salt washes to disrupt low-affinity interactions

• Antibody incubation conditions:

  • Reduce primary antibody concentration

  • Test overnight incubation at 4°C vs. room temperature incubation

  • Add 0.1-0.5% BSA to antibody dilution buffer

• Cross-adsorption:

  • Pre-adsorb antibody with tissues/cells lacking target

  • For tissue work, block endogenous biotin if using biotinylated systems

  • Block endogenous peroxidase/phosphatase for enzyme-based detection

• Sample preparation improvements:

  • Optimize lysis conditions to improve protein solubility

  • For tissues, adjust fixation protocols

  • Remove lipids/reduce background with detergents

What approaches can resolve data inconsistencies when using ERF14 antibodies across different model systems?

When encountering inconsistent results with ERF14 antibodies across different model systems:

• Systematic validation in each model:

  • Verify antibody reactivity in each species/cell type

  • Establish positive/negative controls for each system

  • Consider epitope conservation analysis across species

• Standardize experimental variables:

  • Use consistent sample preparation methods

  • Standardize protein quantification methods

  • Apply identical blocking/washing protocols

• Cross-platform validation:

  • Confirm findings with orthogonal methods

  • Use multiple antibodies targeting different epitopes

  • Complement antibody-based detection with mRNA analysis

• Quantitative considerations:

  • Apply normalization to loading controls appropriate for each system

  • Use calibration standards when comparing absolute levels

  • Consider differences in protein expression levels across systems

• Document experimental conditions:

  • Record antibody lot numbers

  • Document all buffer compositions

  • Note exact timing of each experimental step

How can multiplexing with ERF14 antibodies improve pathway analysis in signaling studies?

Multiplexing strategies with ERF14 antibodies enable comprehensive pathway analysis:

• Antibody selection for multiplexing:

  • Choose antibodies raised in different host species

  • Select conjugated antibodies with non-overlapping fluorophores

  • Validate antibodies specifically for multiplexing applications

• Sequential immunostaining approaches:

  • Apply primary-secondary-stripping cycles

  • Use microwave-based antibody elution between rounds

  • Implement tyramide signal amplification for improved sensitivity

• Advanced multiplexing techniques:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Cyclic immunofluorescence (CycIF) for >10 markers on the same sample

  • Multiplexed ion beam imaging (MIBI) for subcellular resolution

• Analysis considerations:

  • Apply spectral unmixing algorithms for fluorophore separation

  • Implement colocalization analysis for pathway component interactions

  • Use bioinformatics pipelines designed for multiplexed data

This approach enables simultaneous assessment of ERF14 with interacting partners and downstream effectors, providing more comprehensive pathway information than single-marker studies .

What are the critical considerations when using ERF14 antibodies for therapeutic target validation?

When using ERF14 antibodies for therapeutic target validation:

• Antibody functionality assessment:

  • Test for neutralizing vs. non-neutralizing activity

  • Evaluate antibody-dependent cellular cytotoxicity (ADCC) potential

  • Assess complement-dependent cytotoxicity (CDC) capabilities

• Target specificity validation:

  • Confirm on-target activity using genetic knockdown/knockout models

  • Evaluate off-target effects through pathway analysis

  • Assess cross-reactivity with related family members

• Clinically relevant model systems:

  • Validate in patient-derived xenografts or organoids

  • Test in models representing disease heterogeneity

  • Evaluate in systems modeling resistance mechanisms

• Pharmacodynamic considerations:

  Parameter	Assessment Method	Significance
  Target engagement	ELISA, flow cytometry	Confirms binding to intended target
  Functional inhibition	Phosphorylation assays, reporter assays	Verifies biological activity
  Pathway modulation	Transcriptional profiling, proteomics	Demonstrates downstream effects
  Tissue penetration	IHC, imaging	Evaluates biodistribution
  Durability of response	Time-course studies	Informs dosing requirements

• Combination strategies:

  • Test with standard-of-care agents

  • Evaluate synergy with pathway inhibitors

  • Assess potential for resistance development

How do post-translational modifications affect ERF14 antibody epitope recognition?

Post-translational modifications (PTMs) can significantly impact ERF14 antibody epitope recognition through multiple mechanisms:

• Types of PTMs affecting epitope recognition:

  • Phosphorylation: Adds negative charge and can alter epitope conformation

  • Ubiquitination: Large modification that can block antibody access

  • Glycosylation: Can shield epitopes and alter protein folding

  • Acetylation: Modifies charge properties of lysine residues

  • SUMOylation: Large modification affecting protein structure

• Strategies for PTM-specific detection:

  • Use modification-specific antibodies (e.g., phospho-specific)

  • Treat samples with enzymes to remove modifications (phosphatases, deglycosylases)

  • Compare detection before and after modification-inducing treatments

• Experimental verification approaches:

  • Immunoprecipitation followed by mass spectrometry

  • Site-directed mutagenesis of modification sites

  • In vitro modification assays to control PTM status

• Technical considerations:

  • Include phosphatase inhibitors for phospho-epitope preservation

  • Optimize sample preparation to maintain native modifications

  • Consider non-denaturing conditions to preserve conformational epitopes

• Functional impact assessment:

  • Correlate PTM status with protein activity

  • Evaluate subcellular localization changes with modification

  • Assess protein-protein interaction changes dependent on PTM status

How can researchers distinguish true ERF14 signal from technical artifacts?

Distinguishing genuine ERF14 signal from artifacts requires a systematic validation approach:

• Essential controls for validation:

  • Positive controls: Samples known to express ERF14

  • Negative controls: Samples lacking ERF14 expression

  • Technical controls: Secondary antibody only, isotype controls

  • Biological validation: siRNA/shRNA knockdown, CRISPR knockout

• Signal characteristics assessment:

  • Expected subcellular localization

  • Molecular weight confirmation

  • Signal pattern consistency across different antibody lots

  • Correlation with mRNA expression

• Artifact identification checklist:

  Artifact Type	Characteristic Features	Resolution Approach
  Non-specific binding	Multiple unexpected bands, diffuse signal	Optimize blocking, increase washes
  Cross-reactivity	Signals in negative controls	Try alternative antibodies, validate with other methods
  Edge effects (IHC/IF)	Signal concentrated at sample edges	Modify sample preparation, optimize hydration
  Background fluorescence	Diffuse signal across samples	Include autofluorescence controls, use quenching methods
  Fixation artifacts	Inconsistent signal with different fixatives	Compare multiple fixation methods

• Quantification considerations:

  • Apply appropriate background subtraction

  • Use ratio metrics rather than absolute values when possible

  • Implement blinded analysis to remove bias

What approaches help resolve contradictory findings when using different ERF14 antibody clones?

When different ERF14 antibody clones yield contradictory results:

• Epitope mapping analysis:

  • Identify the specific epitopes recognized by each antibody

  • Assess epitope conservation across species and isoforms

  • Evaluate potential for epitope masking in different contexts

• Systematic cross-validation:

  • Test multiple antibody dilutions for each clone

  • Apply identical protocols across all antibodies being compared

  • Evaluate in multiple cell lines/tissue types

• Orthogonal validation methods:

  • Complement antibody detection with mRNA analysis

  • Use tagged-protein expression systems

  • Implement mass spectrometry-based protein detection

• Biological context consideration:

  • Assess cell/tissue-specific post-translational modifications

  • Evaluate protein-protein interactions that may mask epitopes

  • Consider subcellular localization differences

• Reconciliation strategies:

  • Determine if antibodies recognize different isoforms

  • Assess if differences reflect technical vs. biological variance

  • Implement knockout/knockdown controls with each antibody

  • Consider if different antibodies detect different functional states

How should researchers interpret ERF14 antibody data in the context of heterogeneous tissue samples?

Interpreting ERF14 antibody data in heterogeneous tissues requires specialized approaches:

• Cellular heterogeneity assessment:

  • Implement cell type-specific markers in multiplexed staining

  • Use digital pathology tools for quantitative analysis

  • Correlate with single-cell RNA sequencing data when available

• Spatial context analysis:

  • Evaluate expression patterns in relation to tissue architecture

  • Assess gradients of expression across tissue regions

  • Document relationships to stromal components and vasculature

• Quantification strategies:

  • Score both staining intensity and percentage of positive cells

  • Apply histological scoring systems (H-score, Allred score)

  • Use digital image analysis for objective quantification

  • Implement machine learning algorithms for pattern recognition

• Biological interpretation frameworks:

  • Consider developmental context and physiological state

  • Relate expression patterns to known tissue functions

  • Compare with expression of known interaction partners

• Technical considerations:

  • Address tissue sampling bias through multiple sections

  • Standardize fixation and processing procedures

  • Include reference tissues with known expression patterns

  • Implement tissue microarrays for high-throughput comparison

How can ERF14 antibodies be optimized for super-resolution microscopy applications?

Optimizing ERF14 antibodies for super-resolution microscopy requires specific considerations:

• Antibody selection criteria:

  • High specificity and affinity (KD < 10 nM preferably)

  • Low background binding profile

  • Compatibility with sample preparation for super-resolution

• Labeling strategies:

  • Direct conjugation with appropriate fluorophores (Alexa Fluor 647, Atto dyes)

  • Use of smaller detection probes (nanobodies, aptamers, Fab fragments)

  • Site-specific conjugation to maintain antibody orientation

• Sample preparation optimization:

  • Fixation methods preserving ultrastructure (glutaraldehyde)

  • Permeabilization protocols maintaining spatial organization

  • Appropriate blocking to minimize background fluorescence

• Super-resolution specific protocols:

  Technique	Key Optimization Parameters	Special Considerations
  STORM/PALM	Buffer composition (oxygen scavenging system)	Blinking behavior, fluorophore density
  STED	Depletion laser power	Photobleaching resistance, signal strength
  SIM	Sample refractive index matching	Periodic pattern quality, sample thickness
  Expansion microscopy	Expansion factor, protein retention	Epitope accessibility after expansion

• Validation approaches:

  • Correlative imaging with electron microscopy

  • Comparison with known subcellular markers

  • Multi-color imaging with established reference proteins

What are the methodological considerations for developing and validating ERF14 antibodies for liquid biopsy applications?

Developing ERF14 antibodies for liquid biopsy applications presents unique challenges:

• Antibody design considerations:

  • Target secreted or shed forms of ERF14

  • Focus on epitopes stable in circulatory conditions

  • Develop antibodies resistant to proteolytic degradation

  • Consider detection of post-translationally modified forms

• Sample processing optimization:

  • Evaluate plasma vs. serum performance

  • Develop stabilization protocols for pre-analytical phase

  • Standardize centrifugation and storage conditions

  • Assess impact of freeze-thaw cycles on detection

• Assay development parameters:

  • Determine linear dynamic range across physiological concentrations

  • Establish limits of detection and quantification

  • Evaluate matrix effects from blood components

  • Develop spike-in recovery protocols for validation

• Clinical validation approaches:

  • Establish reference ranges in healthy populations

  • Analyze biological variability (diurnal, age, gender)

  • Correlate with tissue expression patterns

  • Compare with existing clinical biomarkers

• Technical implementation:

  • Develop automation-friendly protocols

  • Establish QC measures for clinical laboratory use

  • Create standardized reporting guidelines

  • Implement external quality assessment protocols

How can computational approaches improve ERF14 antibody design and epitope selection?

Advanced computational methods offer powerful tools for ERF14 antibody design:

• Epitope prediction algorithms:

  • B-cell epitope prediction tools (BepiPred, DiscoTope)

  • Molecular dynamics simulations for conformational epitopes

  • Conservation analysis across species for stable epitope regions

  • Identification of regions less subject to post-translational modifications

• Structural biology integration:

  • Homology modeling for epitope accessibility assessment

  • Protein-protein docking simulations

  • Molecular dynamics for flexibility analysis

  • In silico alanine scanning for critical binding residues

• Machine learning applications:

  • Training models on successful vs. failed antibody designs

  • Prediction of cross-reactivity profiles

  • Optimization of physicochemical properties

  • Forecasting of antibody stability and manufacturability

• Workflow integration:

  Computational Step	Output	Experimental Validation
  Epitope prediction	Ranked candidate regions	Peptide binding assays
  Structure modeling	3D visualization of target	Mutagenesis studies
  Antibody modeling	Predicted binding interface	SPR/BLI binding kinetics
  Affinity optimization	Suggested sequence modifications	Directed evolution screening

• Implementation strategies:

  • Pipeline integration with wet lab validation

  • Iterative design-build-test cycles

  • Database development for institutional knowledge retention

  • Integration with high-throughput screening platforms