ETV5 Antibody, Biotin conjugated

What is ETV5 and what biological functions does it regulate?

ETV5 (Ets Variant 5) is a transcription factor belonging to the PEA3 subfamily of ETS domain transcription factors. This protein plays pivotal roles in multiple biological processes including cellular differentiation, proliferation, and migration. ETV5 functions as a regulatory element in several critical pathways:

• Immune regulation: ETV5 controls TH17 cell development and function through STAT3-dependent mechanisms. It directly promotes IL-17A and IL-17F expression by recruiting histone-modifying enzymes to the IL17a-IL17f locus, resulting in increased active histone marks and decreased repressive histone marks . This mechanism creates a feed-forward control of TH17 cytokine production essential for immune responses.

• Reproductive biology: ETV5 is necessary for normal testicular development and spermatogonial stem cell (SSC) maintenance. Studies with Etv5-/- mice demonstrate that while initial testis development appears normal, by 8 days postnatally, SSC density decreases by 17% compared to wild-type controls, progressing to a 32% reduction by day 12 . These findings indicate ETV5's essential role in maintaining the SSC population.

• Oncogenic pathways: ETV5 has been implicated in leukemogenesis, particularly in pre-B acute lymphoblastic leukemia (pre-B-ALL). Certain mutations, especially T505A, can alter gene expression patterns in pre-B cells to promote a leukemic phenotype, primarily by enhancing cellular proliferation .

What is the specificity of biotin-conjugated ETV5 antibodies and how does biotinylation affect their function?

Biotin-conjugated ETV5 antibodies, such as the rabbit polyclonal antibody ABIN1894156, are generated against specific amino acid sequences within the ETV5 protein. This particular antibody recognizes amino acids 8-36 from the N-terminal region of human ETV5 . The specificity is achieved through immunization of rabbits with a KLH-conjugated synthetic peptide corresponding to this sequence.

Biotinylation provides several advantages for antibody applications:

• Enhanced detection sensitivity: The biotin-streptavidin system offers one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), allowing for signal amplification in detection systems.

• Versatility in detection methods: Biotin-conjugated antibodies can be detected using various streptavidin-conjugated reporter molecules (fluorophores, enzymes, gold particles), providing flexibility in experimental design.

• Reduced background: The biotinylation process, when properly controlled, can reduce non-specific binding compared to directly labeled primary antibodies.

• Binding specificity preservation: When biotinylation is performed under optimal conditions, the antibody's ability to recognize its target epitope (AA 8-36 of ETV5) is maintained, as the biotin molecules are typically conjugated to lysine residues away from the antigen-binding site.

It's important to note that the specificity of this antibody has been validated for human ETV5 detection, making it suitable for experiments with human cell lines and tissues .

What applications is the biotin-conjugated ETV5 antibody suitable for?

The biotin-conjugated ETV5 antibody (ABIN1894156) has been validated for several key applications in molecular and cellular biology research:

• Western Blotting (WB): This antibody can detect ETV5 protein in cellular lysates, allowing researchers to quantify expression levels and identify post-translational modifications. The biotin conjugation facilitates detection using streptavidin-HRP systems, providing sensitive chemiluminescent signals .

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody is suitable for both direct and sandwich ELISA formats, enabling quantitative measurement of ETV5 in solution samples .

• Flow Cytometry (FACS): Biotin-conjugated ETV5 antibodies can be used to detect intracellular ETV5 in individual cells, particularly useful for analyzing heterogeneous cell populations such as differentiating T cells or hematopoietic populations .

• Chromatin Immunoprecipitation (ChIP): Though not explicitly validated for ChIP in the product information, similar ETV5 antibodies have been used to study ETV5's direct interaction with target genes. Research has shown that ETV5 directly binds to the IL17a-IL17f locus in TH17 cells, making ChIP a valuable application for ETV5 antibodies in transcriptional research .

The following table summarizes the validated applications and their typical working dilutions:

How should biotin-conjugated ETV5 antibodies be optimally used in flow cytometry protocols?

When using biotin-conjugated ETV5 antibodies for flow cytometry, researchers should follow a carefully optimized protocol to ensure specific detection of intracellular ETV5. The following methodological approach is recommended:

• Cell preparation and fixation:

  • Harvest cells (1-5 × 10^6 cells per sample) and wash twice with PBS

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash twice with PBS containing 1% BSA

• Permeabilization:

  • Permeabilize cells with 0.1-0.5% Triton X-100 or commercial permeabilization buffer for 10 minutes at room temperature

  • For studying ETV5 in T cells, saponin-based permeabilization is recommended as it better preserves cell morphology

• Blocking and antibody staining:

  • Block with 5% normal serum (matching the species of secondary reagent) for 30 minutes

  • Incubate with biotin-conjugated ETV5 antibody (1:100-1:500 dilution) for 45-60 minutes at room temperature or overnight at 4°C

  • Wash three times with permeabilization buffer

• Detection:

  • Incubate with streptavidin-fluorophore conjugate (e.g., streptavidin-PE, streptavidin-APC) for 30 minutes at room temperature

  • Wash three times with permeabilization buffer

  • Resuspend in appropriate buffer for flow cytometric analysis

• Controls:

  • Include an isotype control (biotin-conjugated rabbit IgG) at the same concentration

  • For multicolor panels, include fluorescence minus one (FMO) controls

  • When studying ETV5 variants, wild-type controls should be included

When analyzing T cell subsets, researchers can combine ETV5 detection with surface markers such as CD4 and intracellular markers like RORγt to specifically identify TH17 cells. This approach has been valuable in studies investigating the role of ETV5 in T cell differentiation and function .

What are the optimal conditions for using ETV5 antibodies in Western blotting?

Western blotting with biotin-conjugated ETV5 antibodies requires careful optimization to achieve specific detection with minimal background. The following protocol incorporates best practices based on research applications:

• Sample preparation:

  • Extract total protein from cells or tissues using RIPA buffer supplemented with protease inhibitors

  • For nuclear proteins like ETV5, consider using nuclear extraction protocols to enrich the target

  • Quantify protein concentration using BCA or Bradford assay

  • Prepare 20-50 μg of protein per lane in Laemmli buffer with reducing agent

• Gel electrophoresis and transfer:

  • Separate proteins on 10% SDS-PAGE (ETV5 has a molecular weight of approximately 58 kDa)

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

  • Incubate with biotin-conjugated ETV5 antibody (1:1000-1:5000 dilution) in blocking buffer overnight at 4°C

  • Wash 3 × 10 minutes with TBST

• Detection:

  • Incubate with streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature

  • Wash 3 × 10 minutes with TBST

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or capture using digital imaging system

• Optimization considerations:

  • When detecting endogenous ETV5, longer exposure times may be necessary due to variable expression levels across cell types

  • For difficult-to-detect variants like mutant ETV5 in leukemia cells, sample enrichment through immunoprecipitation may be required prior to Western blotting

  • When analyzing T cells, consider including positive controls such as activated TH1 or TH2 cells, which express higher levels of ETV5

The following table outlines troubleshooting strategies for common Western blotting issues with ETV5 detection:

How can ETV5 antibodies be used in chromatin immunoprecipitation studies to identify direct gene targets?

Chromatin immunoprecipitation (ChIP) using ETV5 antibodies allows researchers to identify direct transcriptional targets of ETV5. While the biotin-conjugated antibody may not be optimal for ChIP due to potential cross-reactivity with endogenous biotin, unconjugated ETV5 antibodies can be effectively employed in ChIP protocols:

• Cross-linking and chromatin preparation:

  • Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Lyse cells and sonicate chromatin to generate 200-500 bp fragments

  • Check sonication efficiency by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads and non-immune IgG

  • Incubate chromatin with ETV5 antibody (2-5 μg) overnight at 4°C

  • Add protein A/G beads and incubate for 2-3 hours

  • Wash extensively to remove non-specific binding

• DNA recovery and analysis:

  • Reverse cross-links and purify DNA

  • Analyze by qPCR with primers targeting suspected ETV5 binding sites

  • For genome-wide analysis, prepare libraries for ChIP-seq

• Target validation considerations:

  • Include positive control regions based on known ETV5 binding sites

  • For TH17 cells, the IL17a-IL17f locus serves as a positive control region

  • Include negative controls (non-target regions) and input controls

  • Validate findings with reporter gene assays or EMSA

When studying ETV5's role in T cells, research has demonstrated that ETV5 directly binds to the IL17a-IL17f locus, recruiting histone-modifying enzymes that alter chromatin structure . This mechanism can be investigated using sequential ChIP (re-ChIP) to detect co-localization of ETV5 with histone modifiers.

ETV5 has been shown to interact with a large number of genomic sites in different cell types, including 10,545 binding sites in type II alveolar cells, 5,378 sites in Ras-transformed mammary gland epithelial cells, and 1,020 sites in mouse embryonic stem cells . This extensive binding profile makes ChIP-seq an essential tool for comprehensive analysis of ETV5 function.

How can ETV5 antibodies be used to investigate T cell differentiation and immune responses?

ETV5 antibodies serve as valuable tools for investigating the role of this transcription factor in T cell differentiation and immune responses. Research has established ETV5 as a critical regulator of TH17 cell development through STAT3-dependent mechanisms . Methodological approaches for studying this process include:

• Analysis of ETV5 expression during T cell differentiation:

  • Isolate naive CD4+ T cells and culture under TH1, TH2, or TH17 polarizing conditions

  • At various time points, assess ETV5 expression by flow cytometry and Western blotting

  • Correlate ETV5 levels with expression of lineage-specific cytokines and transcription factors

  • Research has shown that ETV5 expression is highest in TH1 and TH2 cells compared to other helper cell subsets, yet its functional role is most prominent in TH17 cells

• Functional studies using genetic manipulation:

  • Generate T cell-specific ETV5 knockout models (e.g., Etv5fl/fl CD4-Cre+) to assess the requirement for ETV5 in different T cell subsets

  • Alternatively, use siRNA or CRISPR/Cas9 to knockdown or knockout ETV5 in primary T cells or T cell lines

  • Analyze the impact on cytokine production, proliferation, and differentiation

  • Studies have shown that Etv5-deficient TH17 cells produce significantly less IL-17A compared to control cells, while Etv5-deficient TH2 cells produce more IL-4

• In vivo models to assess ETV5 function in immune responses:

  • Use allergen-induced airway inflammation models (e.g., house dust mite model) to assess the requirement for ETV5 in T cell responses

  • Compare wild-type and Etv5-deficient mice for:

    • Airway inflammation severity

    • Cytokine production in lung tissue and bronchoalveolar lavage fluid

    • T cell infiltration and phenotype

  • Research has demonstrated that mice with Etv5-deficient T cells have reduced airway inflammation and IL-17A/F production in the lung without changes in TH2 cytokine production

The following data table illustrates the impact of ETV5 deficiency on cytokine production by different T helper cell subsets:

These findings highlight the complex and subset-specific roles of ETV5 in regulating T cell cytokine production and function .

What role does ETV5 play in leukemia development and how can antibodies help study these mechanisms?

ETV5 has emerged as a significant factor in leukemogenesis, particularly in pre-B acute lymphoblastic leukemia (pre-B-ALL). ETV5 antibodies can help elucidate the mechanisms by which ETV5 variants contribute to leukemic transformation and progression:

• Identification and characterization of ETV5 mutations in leukemia:

  • Screen leukemia samples for ETV5 variants using sequencing techniques

  • Use ETV5 antibodies to assess expression levels and localization of wild-type and mutant ETV5

  • Compare binding patterns of mutant versus wild-type ETV5 using ChIP assays

  • Recent research has identified several ETV5 variants in leukemias, including R392P, V444I, and T505A mutations

• Functional analysis of ETV5 variants:

  • Express wild-type or mutant ETV5 (R392P, V444I, T505A) in pre-B cell lines

  • Assess the impact on cell proliferation, survival, and differentiation

  • Compare DNA binding capabilities using EMSA or ChIP

  • Research has shown that the T505A ETV5 variant confers a proliferative advantage to pre-B cells, while R392P and V444I affect DNA binding and transcriptional activation

• Mechanistic studies of ETV5-mediated leukemogenesis:

  • Use RNA sequencing to identify genes differentially regulated by ETV5 variants

  • Perform gene set enrichment analysis to identify affected pathways

  • Validate direct targets through ChIP and reporter assays

  • Studies have revealed that T505A ETV5 downregulates the p53 pathway and the anti-proliferative protein BTG2, potentially explaining its pro-leukemic effects

The following table summarizes the functional characteristics of ETV5 variants observed in leukemia:

These findings illustrate how specific ETV5 mutations can alter cellular functions relevant to leukemogenesis, with T505A in particular promoting a proliferative phenotype that may contribute to leukemic transformation .

How can researchers use ETV5 antibodies to study reproductive biology and spermatogonial stem cells?

ETV5 plays a crucial role in reproductive biology, particularly in the maintenance of spermatogonial stem cells (SSCs). ETV5 antibodies can be employed to investigate these functions through various experimental approaches:

• Developmental analysis of ETV5 expression in testicular tissue:

  • Perform immunohistochemistry or immunofluorescence on testicular sections at different developmental stages

  • Use ETV5 antibodies to track expression patterns during testis development

  • Co-stain with markers of spermatogonial stem cells to assess co-localization

  • Studies have shown that while initial testis development appears normal in Etv5-/- mice, by day 8 postnatally, SSC density decreases by 17% compared to wild-type, progressing to a 32% reduction by day 12

• Quantitative assessment of SSC populations:

  • Use flow cytometry with ETV5 antibodies in combination with SSC markers

  • Compare SSC numbers between wild-type and Etv5-deficient models

  • Track SSC loss over time in developmental studies

  • Research has established a method for quantifying SSC density in tubular cross-sections, revealing progressive loss of SSCs in Etv5-/- mice

• In vitro culture systems for mechanistic studies:

  • Isolate SSCs from wild-type and Etv5-deficient mice

  • Compare proliferation, self-renewal, and differentiation capabilities

  • Use ETV5 antibodies to verify knockdown efficiency in siRNA experiments

  • Investigate downstream signaling pathways affected by ETV5 loss

The following table summarizes the progressive loss of spermatogonial stem cells in Etv5-/- mice compared to wild-type controls:

These findings demonstrate that ETV5 is not required for initial SSC development but becomes essential for their maintenance during postnatal development . This progressive loss pattern suggests that ETV5 may regulate factors necessary for SSC niche interactions or self-renewal pathways.

What are common challenges when detecting ETV5 in different cell types and how can they be addressed?

Detecting ETV5 across various cell types presents several technical challenges that require specific troubleshooting approaches:

• Variable expression levels:

  • ETV5 expression varies significantly across cell types, with highest expression reported in TH1 and TH2 cells compared to other T helper subsets

  • For cells with low ETV5 expression, consider:

    • Increasing sample concentration/loading amount

    • Using more sensitive detection systems (e.g., SuperSignal West Femto for Western blots)

    • Employing signal amplification strategies (e.g., tyramide signal amplification for immunostaining)

    • Enriching nuclear fractions for more concentrated ETV5 detection

• Cross-reactivity with other ETS family members:

  • The ETS family contains multiple members with similar domains

  • Validate antibody specificity using:

    • Positive controls (cells known to express ETV5)

    • Negative controls (cells with ETV5 knockdown or knockout)

    • Peptide competition assays

    • Western blot analysis to confirm single band at expected molecular weight

• Detection of ETV5 variants and mutants:

  • Mutations may alter epitope recognition by antibodies

  • When studying ETV5 variants (e.g., R392P, V444I, T505A), verify whether the antibody's target epitope (AA 8-36) is preserved in these variants

  • For comprehensive analysis of wild-type and mutant ETV5, consider using multiple antibodies targeting different epitopes

• Subcellular localization issues:

  • As a transcription factor, ETV5 is predominantly nuclear

  • Ensure proper nuclear permeabilization in flow cytometry and immunostaining:

    • Use appropriate permeabilization reagents (e.g., 0.5% Triton X-100 for fixed cells)

    • Include positive control nuclear markers

    • Consider counterstaining with DAPI or other nuclear dyes

  • For Western blotting, compare cytoplasmic versus nuclear fractions

The following troubleshooting flowchart can guide researchers through ETV5 detection issues:

How can researchers distinguish between wild-type ETV5 and mutant variants in experimental systems?

Distinguishing between wild-type ETV5 and mutant variants requires carefully designed experimental approaches, particularly when studying leukemia-associated mutations such as R392P, V444I, and T505A :

• Antibody-based detection strategies:

  • Most commercially available antibodies, including the biotin-conjugated antibody targeting AA 8-36, recognize both wild-type and mutant ETV5 variants

  • To distinguish variants:

    • Generate mutation-specific antibodies that selectively recognize mutant epitopes

    • Use epitope tagging (e.g., FLAG, HA, or V5) on exogenous wild-type or mutant ETV5 constructs

    • When overexpressing tagged variants, use tag-specific antibodies for detection

• Functional assays to differentiate wild-type from mutant ETV5:

  • DNA binding assays: Electrophoretic mobility shift assay (EMSA) or DNA pulldown assays can assess DNA binding capability. Research has shown that R392P and V444I variants have altered DNA binding capability compared to wild-type ETV5

  • Transcriptional reporter assays: Using ETV5-responsive promoters (e.g., DUSP6 promoter) linked to reporter genes. Studies have demonstrated that R392P and V444I variants show abrogated activation of the DUSP6 promoter

  • Proliferation assays: Measure cell proliferation using methods such as CellTrace Violet dye dilution. T505A ETV5 confers increased proliferation compared to wild-type

• Molecular analysis techniques:

  • RT-PCR followed by sequencing: Design primers flanking mutation sites to amplify and sequence ETV5 transcripts

  • Allele-specific PCR: Design primers that selectively amplify either wild-type or mutant alleles

  • Restriction fragment length polymorphism (RFLP): If mutations create or destroy restriction sites

  • Digital droplet PCR: For precise quantification of wild-type versus mutant ETV5 alleles

• Single-cell analysis approaches:

  • Single-cell RNA-seq: To identify cells expressing wild-type versus mutant ETV5 transcripts

  • Mass cytometry: When combined with metal-conjugated antibodies against downstream targets of ETV5

  • Imaging flow cytometry: To visualize nuclear localization patterns of wild-type versus mutant ETV5

The following table outlines key functional differences that can be used to distinguish between wild-type and mutant ETV5 variants:

These functional differences provide researchers with multiple experimental approaches to distinguish between wild-type and mutant ETV5 variants in their experimental systems .