ETS1 Antibody

What is ETS1 and why are antibodies against it important in research?

ETS1 (ETS proto-oncogene 1) is a transcription factor involved in immune response pathways and cell proliferation regulation. The human version of ETS1 has a canonical length of 441 amino acids and a molecular mass of 50.4 kDa, with 5 identified isoforms . It is primarily localized in the nucleus and cytoplasm, with notable expression in immune tissues including tonsil, spleen, lymph node, bone marrow, and appendix .

ETS1 antibodies are essential tools for studying:

• Immune cell development and function

• Autoimmune disease mechanisms

• Tumor angiogenesis and cancer progression

• Transcriptional regulation in various cellular contexts

These antibodies enable detection and measurement of ETS1 expression, localization, and activity across different experimental systems, making them invaluable for understanding ETS1's diverse biological roles .

What are the major applications for ETS1 antibodies in research?

ETS1 antibodies are versatile tools that support multiple experimental approaches:

The appropriate application depends on your specific research question, with Western blotting being the most commonly validated technique across commercial antibodies .

Which tissue samples are optimal for detecting ETS1 expression?

Based on expression patterns, the following samples provide reliable ETS1 detection:

High Expression Tissues:

• Lymphoid tissues: thymus, spleen, lymph nodes, tonsil

• Bone marrow and appendix

Cell Types with Notable Expression:

• T cells and B cells (quiescent state has higher expression)

• NK cells and NK T cells

• Endothelial cells (during angiogenesis and injury)

Positive Control Samples for Antibody Validation:

• Jurkat cells (human T cell leukemia line)

• Raji cells (B lymphocyte line)

• Human/mouse thymus tissue

• A431 cells (for ICC/IF applications)

Expression levels vary with activation state, particularly in lymphocytes where ETS1 expression decreases upon activation .

What are the key differences between monoclonal and polyclonal ETS1 antibodies?

Select the appropriate antibody type based on your experimental goals and the specific ETS1 region of interest .

How do different antibodies target specific domains or isoforms of ETS1?

ETS1 has distinct functional domains that can be targeted by different antibodies:

Key Domains and Their Functions:

• N-terminal "Pointed/SAM" domain (~80 aa): Protein-protein interactions, ERK2 docking

• Middle transactivation domain: Transcriptional activation

• Autoinhibitory domain: Regulates DNA binding capacity

• C-terminal ETS domain: DNA binding

Isoform Considerations:

• Major isoform (p51/p54): 440/441 amino acids (mouse/human)

• Second isoform (p42): 353/354 amino acids, lacks exon VII (part of autoinhibitory domain)

• Third isoform: 225 amino acids in humans, missing exons III-VI

Antibody Targeting Examples:

• C-4 antibody (Santa Cruz): Targets aa 131-280

• AF7284 (R&D Systems): Targets region Glu127-Val230

• ab225868 (Abcam): Targets C-terminal region (aa 350 to C-terminus)

• Phospho-specific antibodies: Target sites like Ser251

When selecting an antibody, consider which ETS1 domain or isoform is relevant to your research question. Some antibodies may not detect all isoforms depending on their epitope location, which could significantly impact experimental interpretation .

What considerations are important when detecting phosphorylated forms of ETS1?

ETS1 function is regulated through phosphorylation at multiple sites, requiring special considerations for detection:

Key Phosphorylation Sites:

• Threonine 38 (T38): Phosphorylated by ERK2, enhances transcriptional activation

• Serine 41 (S41): Also phosphorylated by ERK2

• Serine 251 (S251): Specific phospho-antibodies available

Methodological Considerations:

• Antibody Selection: Use phospho-specific antibodies (e.g., Anti-Phospho-Ets-1 (S251) Antibody)

• Sample Preparation: Include phosphatase inhibitors in lysis buffers

• Controls:

  • Positive control: Stimulated cells (TGFβ can increase ETS1 phosphorylation)

  • Negative control: Phosphatase-treated samples

• Treatment Conditions: Consider different stimuli that may trigger phosphorylation (VEGFA, TNFα, TGFβ)

Functional Impact of Phosphorylation:

• Affects DNA binding capacity

• Modulates transcriptional activity

• Influences protein-protein interactions

• Regulates ETS1 stability

Phosphorylation state analysis can provide insights into ETS1 activation status rather than just expression levels .

How can ETS1 antibodies be used to study autoimmune disease mechanisms?

ETS1 has been identified as a susceptibility locus for many autoimmune and inflammatory diseases, making it an important target for immunological research .

Methodological Approaches:

• Expression Analysis:

  • Compare ETS1 levels between healthy and autoimmune patient samples using IHC/WB

  • Assess expression in different immune cell populations via flow cytometry

• Functional Studies:

  • Track ETS1 levels during B/T cell activation (expression decreases upon activation)

  • Correlate ETS1 expression with autoantibody production

• Genetic Models:

  • Use antibodies to assess effects of ETS1 variants/polymorphisms

  • Study expression in mouse models like Ets1-/- or Ets1+/-

Key Research Findings:

• ETS1 regulates B and T cell differentiation to limit excessive activation

• ETS1 deficiency leads to development of lupus-like autoimmune disease

• Lyn+/-Ets1+/- mice show greater and earlier production of IgM autoantibodies

• Btk-dependent downregulation of ETS1 is important for normal plasma cell homeostasis

ETS1 antibodies can help elucidate the molecular mechanisms linking ETS1 dysfunction to autoimmunity, potentially identifying new therapeutic targets .

What are the challenges in studying ETS1 in tumor angiogenesis?

ETS1 plays complex roles in tumor angiogenesis, requiring careful experimental design:

Methodological Challenges:

• Cell-type specificity: Distinguishing endothelial-specific ETS1 from tumor cell expression requires careful isolation techniques

• Dual functionality: ETS1 can be both pro- and anti-angiogenic depending on microenvironmental cues

• Dynamic expression: ETS1 is low in resting endothelium but induced during angiogenesis

• Signaling complexity: TGFβ signaling influences ETS1 expression, requiring careful experimental controls

Recommended Approaches:

• Single-cell analysis: Use scRNA-seq to distinguish ETS1 expression in different cell types

• Comparative studies: Immunohistochemical staining comparing tumor vasculature to normal tissue

• Inhibitor experiments: Use pathway inhibitors (e.g., galunisertib for TGFβ) to study regulatory mechanisms

• Knockdown studies: Assess functional effects on endothelial cells with ETS1 suppression

Key Research Findings:

• ETS1 is upregulated in endothelial cells from human tumors compared to normal tissue

• Knockdown of Ets1 inhibits endothelial cell migration and proliferation

• ETS1 upregulation in tumor endothelial cells depends on TGFβ signaling

• TGFβ inhibition can reduce tumor angiogenesis and vascular abnormality in glioblastoma models

Understanding ETS1's role in tumor angiogenesis may lead to new therapeutic strategies targeting the tumor vasculature .

How can ChIP-seq with ETS1 antibodies identify transcriptional targets?

Chromatin immunoprecipitation sequencing (ChIP-seq) with ETS1 antibodies provides insights into direct gene regulation:

Methodological Considerations:

• Antibody Selection:

  • Choose ChIP-validated antibodies

  • Ensure antibody recognizes DNA-binding competent forms of ETS1

  • Consider that autoinhibition affects ETS1's DNA binding capability

• Experimental Controls:

  • Input DNA controls

  • IgG negative controls

  • Positive controls (known ETS1 target regions)

  • Validation with multiple antibodies targeting different epitopes

• Data Analysis Approaches:

  • Motif analysis (ETS1 recognizes core consensus GGAA/T)

  • Integration with expression data

  • Analysis of co-bound transcription factors

  • Pathway enrichment analysis

Research Applications:

• Identification of direct ETS1 transcriptional targets

• Investigation of ETS1 binding to specific promoters (e.g., RPG promoters, id3 promoter)

• Analysis of epigenetic regulation (e.g., ETS1 recruitment of histone deacetylase 1)

• Study of ETS1's role in enhancer activation and chromatin remodeling

Key Published Findings:

• ETS1 binds to ribosomal protein gene (RPG) promoters

• ETS1 recruits histone deacetylase 1 to the id3 promoter

• VEGF stimulation enhances ETS1 chromatin occupancy and acetylation

• ETS1 can act as both activator and repressor depending on context

ChIP-seq provides genome-wide insights into ETS1's direct regulatory targets and mechanisms, complementing other functional approaches .

What positive and negative controls should be used to validate ETS1 antibody specificity?

Proper controls are essential for antibody validation and experimental reliability:

Positive Controls:

Negative Controls:

• Knockdown/Knockout Controls:

  • siRNA-treated cells (documented knockdown efficiency ~80%)

  • shRNA expression systems

  • CRISPR-Cas9 knockout cells

• Antibody Controls:

  • Isotype control antibodies

  • Blocking peptide competition (when available)

  • Primary antibody omission

• Tissue Controls:

  • Low-expressing tissues/cells

  • Species non-reactive samples (if antibody is species-specific)

Validation Methods:

• Multiple antibodies targeting different epitopes

• Multiple detection methods (WB, IHC, IF)

• Band size verification (expected ~50-54 kDa)

• Side-by-side comparison with mRNA expression data

Comprehensive validation ensures confident interpretation of experimental results across different applications .

How should researchers optimize immunohistochemistry protocols for ETS1 detection?

Successful ETS1 immunohistochemistry requires careful protocol optimization:

Antigen Retrieval Methods:

• TE buffer pH 9.0 (primary recommendation for many antibodies)

• Citrate buffer pH 6.0 (alternative method)

• Optimize retrieval time (typically 15-30 minutes)

• Test heat-mediated vs. enzymatic retrieval

Antibody Optimization:

• Titrate antibody concentration:

  • Typical range: 1:50-1:1000 for IHC

  • Start with manufacturer's recommendation

• Test multiple antibodies targeting different epitopes

• Consider using amplification systems for weak signals

Sample Preparation:

• Fixation protocol standardization (duration, fixative type)

• Section thickness (typically 4-6 μm)

• Fresh vs. archived samples (archival may require longer retrieval)

Detection Systems:

• DAB chromogen (brown) with hematoxylin counterstain (used in multiple studies)

• Polymer-based detection systems

• Fluorescent-based multiplex IHC for co-localization studies

Scoring Methods:

• Semi-quantitative scoring (0-3+ intensity)

• Percentage of positive cells

• H-score calculation (intensity × percentage)

• Digital image analysis for objective quantification

Quality Control:

• Include positive controls (tonsil, spleen, thymus)

• Run negative controls (primary antibody omission)

• Include isotype controls

• Document all protocol modifications

Optimization may be required for each tissue type and fixation method to achieve consistent, specific staining .

How can researchers troubleshoot inconsistent Western blot results with ETS1 antibodies?

Western blotting is the most common application for ETS1 antibodies but can present technical challenges:

Sample Preparation Issues:

• Protein Degradation: Use fresh samples and include protease inhibitors

• Phosphorylation Status: Include phosphatase inhibitors if studying phospho-forms

• Extraction Method: Nuclear extraction protocols may be needed (ETS1 is primarily nuclear)

• Loading Control: Ensure consistent loading with appropriate controls (β-actin, GAPDH)

Running Conditions:

• Gel Percentage: 10-12% typically appropriate for 50 kDa ETS1

• Running Buffer: Reducing conditions recommended

• Transfer Method: PVDF membrane commonly used

• Transfer Time: Optimize for complete transfer of 50 kDa proteins

Antibody Conditions:

• Dilution Range: 1:500-1:4000 typical for Western blot

• Incubation Time: Test both 1-hour room temperature and overnight 4°C options

• Blocking Buffer: Optimize (BSA vs. milk, concentration)

• Wash Stringency: Adjust wash buffer composition and duration

Expected Results:

• Band Size: ~50-54 kDa for main isoform

• Multiple Bands: May indicate isoforms (p42 at ~42 kDa) or phosphorylation

• Cell-Type Specific: Expression highest in lymphoid cells and activated endothelial cells

Troubleshooting Steps:

• Test multiple antibodies targeting different epitopes

• Include known positive controls (Jurkat cells, thymus tissue)

• Optimize blocking conditions to reduce background

• Try different secondary antibody detection systems

• Validate antibody specificity with knockdown controls

Following manufacturer's recommended protocols provides a starting point, with optimization required for specific experimental conditions .

How is ETS1 being studied in aging and longevity research?

Recent research has revealed unexpected roles for ETS1 in aging and longevity:

Key Findings:

• ETS1 is downregulated in long-lived individuals (LLIs)

• ETS1 positively regulates ribosomal protein gene (RPG) expression

• ETS1 knockdown reduces RPG expression

• ETS1 knockdown alleviates cellular senescence in human fibroblasts

• Decreased ribosomal biogenesis regulated by ETS1 may be an energy-saving mechanism promoting healthy aging

Methodological Approaches:

• Transcriptome Analysis:

  • Compare ETS1 expression between age groups

  • Analyze co-expression networks with ribosomal genes

• Functional Validation:

  • ETS1 knockdown using shRNA expression plasmids

  • Assessment of effects on cellular senescence markers

  • Measurement of ribosomal biogenesis

• Mechanistic Studies:

  • ChIP-seq to identify ETS1 binding sites across RPG promoters

  • Analysis of chromatin modification patterns

  • Signaling pathway interactions

This emerging area connects ETS1's known roles in transcriptional regulation to novel functions in cellular energy homeostasis and aging, offering potential insights into longevity mechanisms .

How can ETS1 antibodies be used in multiplex imaging approaches?

Advanced multiplex imaging techniques provide spatial context for ETS1 expression:

Multiplex Immunofluorescence:

• Allows simultaneous detection of ETS1 with other markers

• Enables cell type identification and activation state assessment

• Requires careful antibody panel design and spectral unmixing

• Example applications: immune cell subtyping, tumor microenvironment analysis

Multiplex Chromogenic IHC:

• Sequential staining with multiple chromogens

• Allows visualization of ETS1 with other markers on standard microscopes

• Requires optimization of antibody stripping/blocking between rounds

Mass Cytometry Imaging:

• Metal-tagged antibodies for highly multiplexed imaging

• Provides single-cell resolution with dozens of markers

• Requires specialized equipment (CyTOF, Hyperion)

• Ideal for complex immune microenvironment analysis

Spatial Transcriptomics Integration:

• Combine ETS1 protein detection with spatial transcriptomics

• Correlate protein expression with transcriptional programs

• Provides insights into functional states and heterogeneity

Technical Considerations:

• Antibody validation in multiplex context

• Panel design to avoid cross-reactivity

• Signal amplification for low-abundance targets

• Proper controls for each marker

• Advanced image analysis for quantification

These approaches enable studying ETS1 in its native tissue context while preserving spatial relationships with other markers, providing insights into its function in complex microenvironments.

What are the considerations for using ETS1 antibodies in flow cytometry?

Flow cytometry enables quantitative analysis of ETS1 in diverse cell populations:

Sample Preparation:

• Fixation: Paraformaldehyde (typically 2-4%) preserves structure

• Permeabilization: Critical for nuclear factor access

  • Triton X-100 (0.1-0.5%)

  • Saponin (0.1-0.5%)

  • Methanol (90-100%, pre-chilled)

• Buffer composition: PBS with 0.5-2% protein (BSA or FBS)

Antibody Selection:

• Choose flow cytometry-validated antibodies

• Consider directly conjugated antibodies (reduces protocol steps)

• Fluorophore selection based on instrument capabilities and panel design

• Working dilutions typically 1:50-1:100 for flow applications

Controls:

• Isotype controls matched to primary antibody

• FMO (fluorescence minus one) controls

• Positive control samples (e.g., Jurkat cells)

• Biological controls (stimulated vs. unstimulated)

• Knockdown/knockout validation

Panel Design for Context:

• Include lineage markers (e.g., CD3, CD19)

• Add activation markers as needed

• Consider phospho-flow for activation state

• Test for spectral overlap and compensation

Analysis Approaches:

• Define positive populations using appropriate gating

• Measure both percentage positive and mean fluorescence intensity

• Compare expression across different cell subsets

• Analyze changes with stimulation (e.g., TGFβ treatment increases ETS1 in some contexts)

Flow cytometry provides quantitative, single-cell resolution of ETS1 expression across different cell populations, particularly valuable in heterogeneous samples like blood or tumor tissue .

How might emerging antibody technologies advance ETS1 research?

Several cutting-edge antibody technologies show promise for advancing ETS1 research:

Nanobodies and Single-Domain Antibodies:

• Smaller size allows access to hidden epitopes

• Superior tissue penetration

• Potential for improved nuclear localization

• Applications in super-resolution microscopy

• Potential for in vivo imaging

Proximity Ligation Assays:

• Detect protein-protein interactions involving ETS1

• Visualize ETS1 binding partners in situ

• Study complex formation with other transcription factors

• Map signaling networks in different cellular contexts

Highly Multiplexed Protein Detection:

• Mass cytometry (CyTOF) for 40+ parameters simultaneously

• DNA-barcoded antibody technologies

• Sequential fluorescence approaches

• Comprehensive immune cell profiling with ETS1

Antibody Engineering for Functional Studies:

• Intrabodies targeting specific ETS1 domains

• Domain-blocking antibodies for functional inhibition

• Degradation-inducing antibodies (PROTAC approach)

• Conformation-specific antibodies to detect active states

Live-Cell Imaging Applications:

• Cell-permeable antibody fragments

• Genetically encoded antibody-based sensors

• Real-time monitoring of ETS1 localization and activity

• Dynamic studies of ETS1 during cell activation

These emerging technologies will provide more precise tools for studying ETS1 function in complex biological systems, offering new insights into its roles in health and disease.