ETV6 Antibody

What is ETV6 and why is it important in scientific research?

ETV6 (ETS Variant Transcription Factor 6) is a transcriptional repressor that belongs to the ETS family of transcription factors. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and plays essential roles in the development and maintenance of adult hematopoiesis . The importance of ETV6 in scientific research stems from its critical function in normal blood cell development and its involvement in various hematological malignancies. Germ line pathogenic ETV6 variants have been identified in families with predispositions to B-acute lymphoblastic leukemia (B-ALL) and thrombocytopenia, defining a genetic syndrome known as thrombocytopenia 5 (T5) .

Additionally, ETV6 is implicated in various types of cancers beyond leukemia, including breast and prostate cancer, making it a significant target for therapeutic interventions . Studies have shown that ETV6 functions primarily as a transcriptional repressor, binding to specific DNA sequences and recruiting co-repressors to inhibit gene expression. This repressive activity is crucial for proper blood cell development in the bone marrow . Understanding ETV6's function and regulation is therefore essential for advancing our knowledge of both normal hematopoiesis and cancer biology.

What are the common applications of ETV6 antibodies in laboratory research?

ETV6 antibodies serve multiple crucial functions in laboratory research, with applications spanning from protein detection to complex genomic analyses. Western blot (WB) analysis represents one of the most common applications, allowing researchers to detect and quantify ETV6 protein expression in various cell types and tissues . The recommended dilution range for Western blot applications is typically 1:500 to 1:2000, depending on the specific antibody and experimental conditions .

Beyond protein detection, ETV6 antibodies are essential tools for chromatin immunoprecipitation studies, including advanced techniques such as CUT&RUN (Cleavage Under Targets and Release Using Nuclease) . These approaches enable genome-wide mapping of ETV6 binding sites, providing insights into its transcriptional regulatory network. ETV6 antibodies are also utilized in ELISA applications for quantitative protein detection, and in immunohistochemistry studies to visualize ETV6 localization in tissue sections .

In functional genomics research, ETV6 antibodies help validate gene silencing efficiency in knockdown experiments, such as those involving shRNA screens designed to identify ETV6 modulators . These antibodies are invaluable for investigating how various experimental conditions or genetic manipulations affect ETV6 expression, localization, and function, contributing to our understanding of ETV6's role in normal development and disease states.

What characteristics should researchers look for when selecting an ETV6 antibody?

When selecting an ETV6 antibody for research applications, several critical characteristics must be considered to ensure experimental success. First, antibody specificity is paramount—the selected antibody should recognize ETV6 with high specificity and minimal cross-reactivity with other proteins, especially other ETS family members that share structural similarities . Validation data demonstrating the antibody's specificity through multiple techniques should be evaluated.

Species reactivity is another crucial consideration. Many ETV6 antibodies, such as the CAB15668 polyclonal antibody, offer cross-reactivity across human, mouse, and rat samples, providing flexibility for researchers working with different model systems . For researchers studying specific ETV6 variants or mutations, it is essential to verify that the antibody recognizes the region of interest and can distinguish between wild-type and mutant forms if necessary.

The antibody's performance in intended applications must be thoroughly validated. For instance, an antibody intended for Western blot analysis should demonstrate clear, specific bands at the expected molecular weight in positive control samples such as HepG2 cells, mouse lung, mouse spleen, or rat testis . Additionally, researchers should consider the antibody's demonstrated performance in specialized applications like CUT&RUN assays, which may require particular binding characteristics .

Finally, technical specifications such as antibody isotype, host species, immunogen sequence, and recommended dilutions should align with experimental designs and laboratory protocols. The form of the antibody (unconjugated or conjugated with a detection molecule) should be appropriate for the planned application . Thorough evaluation of these characteristics will help ensure selection of an appropriate ETV6 antibody for specific research needs.

How can researchers effectively use ETV6 antibodies in CUT&RUN assays?

The CUT&RUN (Cleavage Under Targets and Release Using Nuclease) assay represents a powerful technique for mapping ETV6 binding sites across the genome with high resolution and low background. Successfully implementing this method with ETV6 antibodies requires careful consideration of several technical aspects. Based on published protocols, researchers have effectively performed CUT&RUN assays using various cell numbers depending on cell type: one million HPC5 cells, 50,000-100,000 LSK cells, or 500,000 CD34+ cells . This flexibility in input material makes the technique adaptable to rare cell populations relevant to ETV6 research.

When selecting antibodies for CUT&RUN assays, specificity is critical. Researchers should use a validated anti-ETV6 antibody along with appropriate isotype control antibodies to distinguish specific from non-specific binding events . For instance, normal rat immunoglobulin G isotype control antibody (#2729, Cell Signaling Technologies) has been successfully used as a control in ETV6 CUT&RUN experiments . The binding characteristics of the antibody are particularly important for this application, as the antibody must recognize the native conformation of ETV6 bound to chromatin.

For optimal results, researchers should consider adapting established protocols to their specific experimental setup. The basic workflow involves binding the primary antibody to the target in permeabilized cells, followed by addition of protein A/G-MNase fusion protein, activation of calcium-dependent nuclease activity, and isolation of released DNA fragments for sequencing. Integration of CUT&RUN data with other genomic approaches, such as ATAC-seq, Hi-C, and RNA-seq, can provide comprehensive insights into ETV6's role in transcriptional regulation . This multi-omics approach has been particularly valuable for identifying novel ETV6 targets, including genes involved in inflammatory signaling.

What approaches are recommended for studying ETV6 variant effects on protein function?

Investigating the functional consequences of ETV6 variants requires a multi-faceted approach to comprehensively assess their impact on protein function. In vitro studies have revealed that pathogenic ETV6 variant proteins exhibit impaired repressor activity, reduced DNA binding, and abnormal subcellular localization . To evaluate these effects, researchers can employ a range of complementary techniques targeting different aspects of ETV6 function.

For assessing transcriptional repressor activity, reporter gene assays using ETV6-responsive promoters provide valuable insights. For example, the EBS3tk promoter system, which contains three tandem ETV6 binding sites followed by the HSV-TK promoter, has been successfully used to measure wild-type and variant ETV6 repressor function . By comparing the expression levels of reporter genes in cells expressing wild-type versus variant ETV6, researchers can quantify the impact of specific variants on transcriptional repression.

DNA binding capacity can be evaluated through techniques such as electrophoretic mobility shift assays (EMSAs) or more comprehensive genomic approaches like CUT&RUN . These methods allow researchers to determine whether variants affect ETV6's ability to recognize and bind its target DNA sequences. For subcellular localization studies, fluorescently tagged ETV6 constructs or immunofluorescence using ETV6 antibodies can reveal whether variants alter the protein's normal nuclear localization .

Protein-protein interaction analyses, including co-immunoprecipitation experiments, are essential for investigating whether variants affect ETV6's ability to dimerize or interact with co-repressors. This is particularly important as some missense variants retain the ability to dimerize with wild-type ETV6, potentially exerting dominant-negative effects . Integrating these experimental approaches provides a comprehensive understanding of how specific variants impact different aspects of ETV6 function, contributing to our knowledge of variant pathogenicity mechanisms.

How should researchers design shRNA screens to identify ETV6 modulators?

Designing effective shRNA screens to identify modulators of ETV6 activity requires careful consideration of cellular systems, readout strategies, and validation approaches. A successful example of such a screen was implemented using a modified Reh pre-B ALL cell line that lacks endogenous ETV6 expression due to a t(12;21)(p13;q22) translocation and 12p13 deletion . This cellular system was engineered to express a Blasticidin-resistance gene (BlastR) under the control of an artificial ETV6-responsive promoter (EBS3tk), creating a direct readout of ETV6 transcriptional repression activity .

The key components of this system included: (1) a reporter gene (BlastR) expressed through an ETV6-responsive promoter; (2) reintroduction of wild-type ETV6 through lentiviral transduction to establish baseline repression; and (3) a genome-wide shRNA library to systematically knock down potential ETV6 modulators . Successful implementation required initial validation of the reporter system, demonstrating that ectopically expressed ETV6 significantly reduced BlastR expression compared to control clones (2.7-fold reduction, p ≤ 0.001) .

For screening, researchers should establish robust positive and negative controls. In the published screen, a reduction in ETV6 repressive function would lead to increased BlastR expression and enhanced cell survival under blasticidin selection, creating a selectable phenotype for identifying potential modulators . Post-screening validation is crucial and should include: (1) confirmation of target gene knockdown efficiency by qPCR or RNA-targeted sequencing; (2) assessment of the impact on multiple ETV6 target genes, not just the reporter; and (3) orthogonal validation using independent shRNAs or complementary approaches .

Through this systematic approach, researchers identified 13 shRNAs that efficiently knocked down their target genes and impaired ETV6 transcriptional activity, with five genes (AKIRIN1, COMMD9, DYRK4, JUNB, and SRP72) emerging as particularly influential ETV6 modulators . This demonstrates the power of well-designed shRNA screens for uncovering novel components of transcriptional regulatory networks.

What are the challenges in distinguishing wild-type ETV6 from variant forms using antibody-based techniques?

Distinguishing between wild-type ETV6 and variant forms using antibody-based techniques presents several technical challenges that researchers must address for accurate experimental outcomes. The primary challenge is antibody epitope specificity—many commercial antibodies are raised against specific regions of the ETV6 protein, which may or may not encompass the variant site of interest . For instance, antibodies targeting the N-terminal region (amino acids 1-300) may not distinguish variants in the ETS DNA-binding domain, such as the R355X variant described in the literature .

Another challenge is that some ETV6 variants result in altered protein expression levels rather than structural changes. For example, germline ETV6 variants linked to leukemia predisposition can lead to impaired transcription repressor activity and altered nuclear localization . In such cases, quantitative techniques like Western blotting may detect differences in expression levels, but qualitative differences in protein function would remain undetected without functional assays.

Researchers can address these challenges through several approaches: (1) using antibodies targeting different epitopes to comprehensively assess protein expression and localization; (2) combining antibody-based detection with functional assays that directly measure ETV6 activity; (3) employing recombinant systems with epitope tags to distinguish wild-type from variant proteins; and (4) integrating genomic approaches like CUT&RUN to assess functional differences in DNA binding between wild-type and variant ETV6 . These multifaceted approaches provide more comprehensive insights than relying solely on antibody-based detection methods.

What controls are essential when using ETV6 antibodies in Western blot applications?

Implementing appropriate controls in Western blot applications using ETV6 antibodies is critical for ensuring reliable and interpretable results. Positive control samples with known ETV6 expression should be included in every experiment. According to product specifications, validated positive control samples for ETV6 detection include HepG2 cells, mouse lung tissue, mouse spleen tissue, and rat testis . These controls confirm the antibody's ability to recognize the target protein in the experimental system and provide reference points for evaluating signal intensity.

Negative controls are equally important for assessing antibody specificity. These may include samples known to lack ETV6 expression or samples where ETV6 has been knocked down using shRNA or CRISPR-Cas9 techniques. The Reh pre-B ALL cell line, which lacks endogenous ETV6 expression due to a t(12;21)(p13;q22) translocation and 12p13 deletion, represents an excellent negative control for ETV6 antibody validation . Additionally, isotype control antibodies matching the host species and immunoglobulin class of the ETV6 antibody should be included to identify non-specific binding.

Loading controls must be carefully selected to normalize for differences in protein loading across samples. Conventional loading controls like GAPDH or β-actin are appropriate for most applications, but nuclear protein markers such as histone H3 or lamin B1 may be more suitable given ETV6's nuclear localization . When studying ETV6 variants, wild-type ETV6 expression constructs should be included as controls to directly compare protein expression, molecular weight, and antibody recognition.

Researchers should also validate antibody specificity through peptide competition assays, where pre-incubation of the antibody with its immunizing peptide should abolish specific binding. Finally, concentration gradients of recombinant ETV6 protein can serve as standards for quantitative assessments. Adhering to these control measures ensures robust and reproducible Western blot results when working with ETV6 antibodies.

What are the recommended approaches for validating ETV6 antibody specificity across different species?

Validating ETV6 antibody specificity across different species requires a systematic approach to ensure reliable cross-species reactivity and experimental reproducibility. While many commercial antibodies claim cross-reactivity with human, mouse, and rat ETV6 , independent validation is essential before conducting cross-species comparative studies. The first step in this validation process is sequence analysis—researchers should compare the amino acid sequence of the immunogen used to generate the antibody with the corresponding sequences in target species. Higher sequence homology increases the likelihood of cross-reactivity.

Experimental validation should begin with Western blot analysis using positive control samples from each species of interest. For ETV6 antibodies, appropriate positive controls include HepG2 cells for human samples, mouse lung or spleen tissue for murine studies, and rat testis for rat experiments . Clean, specific bands at the expected molecular weight in each species provide preliminary evidence of cross-reactivity.

For more rigorous validation, researchers should include samples with manipulated ETV6 expression levels. Comparing wild-type samples to those with ETV6 knockdown or knockout should demonstrate corresponding decreases in antibody signal intensity. Similarly, overexpression systems can confirm the antibody's ability to detect increased ETV6 levels. Importantly, these manipulation experiments should be conducted in cell lines or tissue samples from each species of interest.

When validating antibodies for specialized applications like CUT&RUN assays, which require recognition of the native, chromatin-bound conformation of ETV6, additional controls are necessary . These include isotype control antibodies and comparison of binding profiles to established ETV6 binding patterns or motifs. Cross-validation with multiple antibodies targeting different epitopes of ETV6 can provide further confidence in specificity across species. Through this comprehensive validation approach, researchers can ensure reliable performance of ETV6 antibodies in cross-species studies.

What modifications to standard protocols are needed when using ETV6 antibodies for immunoprecipitation?

Optimizing immunoprecipitation (IP) protocols for ETV6 requires specific modifications to standard procedures due to the protein's nuclear localization and involvement in protein complexes. Nuclear extraction protocols must be carefully optimized to efficiently release ETV6 from chromatin while preserving protein-protein interactions. A two-step extraction process is often beneficial: first using a mild buffer to extract cytoplasmic proteins, followed by a nuclear extraction buffer containing appropriate salt concentrations (typically 300-420 mM NaCl) to release nuclear proteins while maintaining antibody-antigen binding capacity.

Cross-linking steps may be necessary when studying ETV6's interactions with chromatin or other nuclear proteins. A brief formaldehyde fixation (1% formaldehyde for 10 minutes at room temperature) before cell lysis can stabilize protein-DNA and protein-protein interactions that might otherwise be disrupted during extraction. For co-immunoprecipitation studies investigating ETV6's interactions with other proteins, gentler lysis conditions and lower salt concentrations may be required to preserve protein complexes.

The choice of antibody amount and incubation conditions is critical for successful ETV6 immunoprecipitation. Typically, 2-5 μg of antibody per 500 μg of nuclear extract yields good results, with overnight incubation at 4°C providing optimal antigen binding. When selecting protein A/G beads for capturing the antibody-antigen complex, pre-clearing the lysate with beads alone before adding the antibody can reduce non-specific binding.

Washing conditions must balance removing non-specific interactions while retaining specific ETV6 complexes. A series of washes with decreasing salt concentration is often effective. For elution, gentle methods such as competitive elution with immunizing peptide may better preserve complex integrity than harsh denaturing conditions. Finally, when analyzing results, controls should include isotype-matched control antibodies and input samples to accurately assess enrichment. These protocol modifications enhance the specificity and efficiency of ETV6 immunoprecipitation, enabling more reliable studies of its interactions and functions.

How can ETV6 antibodies be used to study leukemia models with ETV6 variants?

ETV6 antibodies serve as essential tools for investigating leukemia models harboring ETV6 variants, enabling researchers to elucidate the molecular mechanisms underlying leukemogenesis. Immunoblotting represents a fundamental application, allowing quantitative comparison of ETV6 protein levels between wild-type and variant-expressing cells . This approach has revealed that some pathogenic variants affect protein stability or expression levels, while others primarily impact functional properties despite normal expression. When conducting such comparisons, researchers should normalize ETV6 levels to appropriate loading controls and include multiple biological replicates to account for variability.

Immunofluorescence microscopy using validated ETV6 antibodies provides critical insights into the subcellular localization of variant proteins. Studies have demonstrated that pathogenic ETV6 variants can exhibit abnormal subcellular localization, which contributes to their functional impairment . By combining ETV6 immunostaining with markers for nuclear and cytoplasmic compartments, researchers can quantitatively assess localization defects that may contribute to disease pathogenesis.

For genome-wide binding analyses, ETV6 antibodies enable techniques such as CUT&RUN to map binding sites of both wild-type and variant proteins . This approach has revealed that the R355X variant, associated with thrombocytopenia and leukemia predisposition, exhibits altered genome-wide binding patterns, particularly at inflammatory gene loci such as TNF . Integration of binding data with transcriptomic analyses provides comprehensive insights into how altered ETV6 binding affects gene expression programs in leukemic cells.

Importantly, when studying dominant-negative variants that retain dimerization with wild-type ETV6, researchers may need specialized approaches to distinguish the behavior of variant-wild-type heterodimers from wild-type homodimers . Sequential immunoprecipitation using antibodies recognizing different epitopes or tagged protein versions can help dissect the complex interactions and functional consequences of these variants in leukemia models.

What insights can be gained from studying ETV6 binding patterns in different hematological conditions?

Comprehensive analysis of ETV6 binding patterns across different hematological conditions provides fundamental insights into disease-specific transcriptional dysregulation. Studies using CUT&RUN and related techniques have revealed that ETV6 binds to inflammatory gene loci, including TNF, in hematopoietic stem and progenitor cells (HSPCs) . This binding is functionally significant, as ETV6 represses the expression of these inflammatory genes during stress hematopoiesis. Aberrant regulation of this inflammatory program due to ETV6 mutations or alterations may contribute to hematological disorders by disrupting normal HSPC function.

In leukemia with germline ETV6 variants, whole-transcriptome and whole-genome sequencing have uncovered a profound influence of these variants on the leukemia transcriptional landscape . Remarkably, different ALL subsets associated with ETV6 variants invoke unique patterns of somatic cooperating mutations. Approximately 70% of ALL cases with damaging germline ETV6 variants exhibit hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11 . The remaining 30% of cases have diploid leukemia genomes with a high frequency of somatic copy-number loss of PAX5 and ETV6, displaying gene expression patterns that strikingly mirror those of ALL with somatic ETV6-RUNX1 fusion .

This heterogeneity in genomic and transcriptomic profiles suggests that different ETV6 variants may predispose to distinct leukemia subtypes through specific molecular mechanisms. Furthermore, the observation that certain ETV6 germline variants can give rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes, indicates that the hematopoietic cell context significantly influences how ETV6 dysfunction manifests as disease .

Studying these binding patterns and associated transcriptional networks across different hematological conditions not only enhances our understanding of disease pathogenesis but also identifies potential therapeutic targets. The repression of inflammatory genes by ETV6, particularly in stress conditions, represents a critical regulatory mechanism that, when disrupted, may contribute to leukemogenesis through enhanced inflammatory signaling.

How might single-cell approaches enhance our understanding of ETV6 function in heterogeneous cell populations?

Single-cell approaches offer revolutionary potential for dissecting ETV6 function in complex tissues and heterogeneous cell populations, overcoming limitations of bulk analysis techniques. Traditional bulk methods average signals across populations, potentially masking critical cell-specific ETV6 activities. Single-cell RNA sequencing (scRNA-seq) enables researchers to identify cell-type-specific transcriptional programs regulated by ETV6, revealing how ETV6 dysfunction differentially affects distinct cell populations within the hematopoietic hierarchy . This approach has been successfully integrated with other techniques to characterize ETV6's role in hematopoietic stem and progenitor cells (HSPCs) and could reveal how ETV6 variants differentially impact various cell populations.

Emerging techniques like single-cell CUT&RUN or single-cell ATAC-seq provide unprecedented resolution for mapping ETV6 binding sites or chromatin accessibility changes in rare cell populations. These approaches could reveal cell-type-specific binding patterns of ETV6 that may be diluted or missed entirely in bulk analyses. For instance, investigating how ETV6 binding patterns differ between stem cells and committed progenitors could illuminate its role in lineage commitment decisions and explain why certain ETV6 variants predispose to specific hematological malignancies.

Single-cell protein analysis techniques such as mass cytometry (CyTOF) or single-cell Western blotting, when performed using validated ETV6 antibodies, could quantify ETV6 protein levels and post-translational modifications at the single-cell level. This would enable correlation of ETV6 expression or modification states with cell phenotypes or developmental trajectories. For example, correlating TNF expression with ETV6 levels in individual cells could provide direct evidence for ETV6's repressive function in regulating inflammatory gene expression .

Perhaps most powerfully, integrated single-cell multi-omics approaches that simultaneously profile transcriptomes, proteomes, and epigenomes in the same cells could provide comprehensive understanding of how ETV6 coordinates multiple levels of cellular regulation. These emerging technologies offer unprecedented opportunities to dissect the complex roles of ETV6 in normal development and disease states at previously unattainable resolution.

What novel antibody-based technologies might advance ETV6 research in the near future?

Emerging antibody-based technologies promise to significantly expand our capabilities for studying ETV6 biology and its role in disease pathogenesis. Proximity ligation assays (PLA) represent one such technology with potential to revolutionize our understanding of ETV6 protein interactions. By combining antibody recognition with DNA amplification techniques, PLA enables visualization and quantification of protein-protein interactions with single-molecule sensitivity in situ. This approach could provide spatial information about ETV6 interactions with cofactors in different nuclear compartments, offering insights into how pathogenic variants disrupt specific protein complexes in their native cellular context.

CRISPR-based tagging systems combined with antibody detection offer another promising approach. By integrating small epitope tags into endogenous ETV6 using CRISPR-Cas9, researchers can study the native protein using highly specific anti-tag antibodies without overexpression artifacts. This strategy enables live-cell imaging of ETV6 dynamics and could be particularly valuable for investigating how ETV6 localization and chromatin association change during differentiation or in response to cellular stress.

Advanced imaging techniques like super-resolution microscopy combined with highly specific ETV6 antibodies could reveal previously undetectable details about ETV6's nuclear organization and association with transcriptional hubs. For instance, techniques such as Stimulated Emission Depletion (STED) microscopy or Stochastic Optical Reconstruction Microscopy (STORM) could visualize ETV6 distribution within the nucleus at nanometer-scale resolution, potentially identifying distinct subnuclear compartments where ETV6 exerts its repressive function.

Mass spectrometry-based techniques coupled with antibody-mediated enrichment represent another frontier. Approaches like Selective Isolation of Labeled Entities by Antibodies (SILAC) combined with ETV6 immunoprecipitation could identify dynamic changes in ETV6 protein complexes under different conditions or in the presence of pathogenic variants. These technologies will collectively advance our understanding of ETV6 biology at unprecedented resolution, potentially revealing new therapeutic opportunities for ETV6-associated diseases.