ETV5 Antibody, FITC conjugated

What is ETV5 and why is it important in research?

ETV5 (ETS variant transcription factor 5, also known as ERM) is a member of the ETS oncogene family that binds to DNA sequences containing the consensus nucleotide core sequence 5'-GGAA-3' . It functions primarily as a transcription factor and is required for spermatogonial stem cell self-renewal . ETV5 plays a critical role in branching morphogenesis during kidney development and can be negatively regulated by COP1, a tumor suppressor . Recent studies have identified ETV5 as a prognostic marker in multiple cancers, showing abnormally high expression levels in 25 tumor types compared to adjacent normal tissues . Its molecular weight is approximately 58-60 kDa (510 amino acids) . Understanding ETV5 expression and function is essential for researching developmental processes, stem cell biology, and cancer pathogenesis.

What are the key technical specifications for ETV5 antibodies?

ETV5 antibodies are available in various formats, including monoclonal and polyclonal variants. Typical specifications include:

For FITC-conjugated variants specifically, researchers should note that the fluorescein conjugation occurs typically at lysine residues and may affect binding in applications requiring the labeled region .

How does FITC conjugation affect antibody functionality and storage?

FITC (fluorescein isothiocyanate) conjugation adds a fluorescent tag to the antibody, enabling direct visualization without secondary antibodies. When using FITC-conjugated ETV5 antibodies, researchers should consider:

• The conjugation may reduce antibody binding affinity by 10-15% compared to unconjugated variants due to potential steric hindrance or modification of critical binding domains.

• FITC-conjugated antibodies are typically more sensitive to photobleaching and require storage in dark conditions at 4°C (not -80°C like unconjugated variants).

• The FITC fluorophore has excitation/emission wavelengths of 495/519nm, which may overlap with cellular autofluorescence in some tissue types, particularly those rich in flavin coenzymes or NADH.

• FITC conjugation generally maintains the nuclear localization pattern observed for ETV5 in immunofluorescence applications, consistent with its function as a transcription factor .

What are the optimal protocols for immunofluorescence using FITC-conjugated ETV5 antibodies?

For successful immunofluorescence with FITC-conjugated ETV5 antibodies, the following protocol shows optimal results based on published methods:

• Fixation: 4% paraformaldehyde at room temperature for 15 minutes preserves ETV5 antigenicity while maintaining cellular structure .

• Permeabilization: 0.1-0.3% Triton X-100 for 10 minutes allows antibody access to the nuclear compartment where ETV5 predominantly localizes.

• Blocking: 5% normal serum (matched to secondary antibody host if using unconjugated primary) or 3-5% BSA for 1 hour reduces non-specific binding.

• Primary antibody incubation: Dilution ratios of 1:250 to 1:500 typically provide optimal signal-to-noise ratio for FITC-conjugated ETV5 antibodies.

• Nuclear counterstaining: DAPI or Hoechst at standard concentrations, but avoid propidium iodide which may spectrally overlap with FITC.

For comparison, when using unconjugated ETV5 antibodies, studies have reported successful staining with a 1:500 dilution followed by fluorophore-conjugated secondary antibodies .

How should I design experiments to study ETV5 in embryonic stem cells?

When studying ETV5 in embryonic stem cells (ESCs), consider the following experimental design elements:

• Context: ETV5 expression is regulated by OCT3/4 in ESCs and plays a role in cellular proliferation and differentiation .

• Functional redundancy: Design experiments considering that ETV4 and ETV5 show functional redundancy; double knockout studies may be necessary to observe phenotypic effects .

• Target genes: Include analysis of stem cell-related genes that are downstream of ETV5, such as Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, which are significantly repressed in Etv4/5 double-knockout ES cells .

• Differentiation assays: Monitor ectoderm marker genes (Fgf5, Sox1, Pax3) during embryoid body formation, as ETV5 affects their expression during differentiation .

• Controls: Include appropriate controls such as:

  • Wild-type ESCs

  • Single ETV4 or ETV5 knockout cells

  • Rescue experiments with artificial expression of ETV4/ETV5

For immunofluorescence studies specifically, nuclear counterstaining is essential to confirm the nuclear localization of ETV5 in stem cell populations .

What are the key considerations for using FITC-conjugated ETV5 antibodies in flow cytometry?

When performing flow cytometry with FITC-conjugated ETV5 antibodies, researchers should address these important technical considerations:

• Fixation and permeabilization: Since ETV5 is primarily nuclear, use fixation agents like 4% paraformaldehyde followed by permeabilization with methanol or saponin-based permeabilization buffers to allow antibody access to the nucleus.

• Compensation settings: FITC emission spectrum overlaps with PE; proper compensation controls are essential when multiplexing with other fluorophores.

• Titration: Perform antibody titration (typically testing 1:50 to 1:500 dilutions) to determine optimal concentration for signal-to-noise ratio in your specific cell type.

• Controls: Include:

  • Isotype control (FITC-conjugated isotype-matched irrelevant antibody)

  • Unstained cells

  • Cells with known high and low ETV5 expression

  • FMO (fluorescence minus one) controls when multiplexing

• Gating strategy: Since ETV5 is expressed at varying levels across tumor and normal tissues, establish clear gating strategies based on control populations .

For cancer studies specifically, researchers should note that ETV5 expression varies significantly across tumor types, with 25 types showing significantly higher expression compared to normal tissues .

How can FITC-conjugated ETV5 antibodies be used in cancer research?

FITC-conjugated ETV5 antibodies offer valuable tools for cancer research applications:

• Prognostic marker analysis: Recent studies identify ETV5 as a prognostic marker in multiple cancers, with abnormally high expression correlating with unfavorable outcomes . FITC-conjugated antibodies enable rapid screening of tissue samples.

• Drug sensitivity studies: ETV5 expression has been linked to differential drug sensitivity in multiple malignancies . Researchers can use FITC-conjugated ETV5 antibodies to:

  • Identify ETV5-expressing tumors via flow cytometry or immunofluorescence

  • Correlate expression levels with response to therapeutic agents

  • Monitor changes in ETV5 expression during treatment

• Epigenetic modification research: ETV5 expression is associated with epigenetic modulators like EZH2 . FITC-conjugated antibodies can be combined with probes for epigenetic marks to analyze their spatial relationship within tumor samples.

• Subcellular localization: While predominantly nuclear, ETV5 localization patterns may vary in different cancer types. FITC-conjugated antibodies allow direct visualization of any altered localization patterns .

• Cancer stem cell identification: Given ETV5's role in stem cell maintenance, FITC-conjugated antibodies can help identify cancer stem cell populations within heterogeneous tumors .

Recent research indicates that in hepatocellular carcinoma, ETV5 expression correlates with tumor pathological stage and adverse patient outcomes, making it a particularly valuable target for investigation in liver cancer research .

What methodologies are recommended for studying ETV5's relationship with epigenetic modifiers?

To investigate the relationship between ETV5 and epigenetic modifiers like EZH2, consider these methodological approaches:

• Co-immunoprecipitation with FITC detection: Use unconjugated anti-ETV5 for pulldown followed by FITC-conjugated antibodies to detect interaction partners in Western blot or microscopy.

• ChIP-seq analysis: Chromatin immunoprecipitation using ETV5 antibodies can identify genomic binding sites, which can be correlated with histone modification patterns established by epigenetic modifiers like EZH2.

• Proximity ligation assay (PLA): Combine FITC-conjugated ETV5 antibodies with antibodies against epigenetic modifiers to visualize protein-protein interactions in situ with subcellular resolution.

• FRET/FLIM analysis: When paired with appropriate complementary fluorophore-tagged antibodies against epigenetic modifiers, FITC-conjugated ETV5 antibodies can be used for Förster resonance energy transfer studies.

• Sequential ChIP (re-ChIP): This technique can determine if ETV5 and epigenetic modifiers co-occupy the same genomic regions.

Recent studies demonstrate that ETV5 facilitates cell proliferation and reduces sensitivity to the EZH2 inhibitor GSK126 via regulating EZH2 . This finding suggests that the ETV5-EZH2 axis represents a promising target for epigenetic therapies.

How can I optimize dual immunofluorescence protocols to study ETV5 interactions with other proteins?

For optimal dual immunofluorescence to study ETV5 interactions with other proteins:

• Antibody selection:

  • Choose primary antibodies raised in different host species

  • For FITC-conjugated ETV5 antibody, select complementary fluorophores with minimal spectral overlap (e.g., Cy5, Alexa 647)

  • Confirm that epitopes of interaction partners are not masked by protein-protein interaction

• Sequential staining protocol:

  • Fix cells with 4% paraformaldehyde (15 minutes, room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 (10 minutes)

  • Block with 3-5% BSA (1 hour)

  • Incubate with FITC-conjugated ETV5 antibody (1:250-1:500, overnight at 4°C)

  • Wash thoroughly (3x PBS)

  • Incubate with unconjugated primary antibody against interaction partner

  • Wash thoroughly (3x PBS)

  • Incubate with fluorophore-conjugated secondary antibody

  • Counterstain nucleus and mount

• Controls:

  • Single antibody controls to assess bleed-through

  • Competition assays using excess unconjugated antibodies

  • Colocalization with known interaction partners as positive controls

When studying ETV5's interactions with EZH2 specifically, researchers should note that while ETV5 is primarily nuclear, the specific subnuclear domains where these interactions occur may require super-resolution microscopy techniques for proper visualization .

What are common issues when using FITC-conjugated ETV5 antibodies and how can they be resolved?

For ETV5 specifically, researchers should be aware that fixation methods significantly impact nuclear antigen detection. The recommended 4% paraformaldehyde fixation for 15 minutes has been validated for ETV5 detection in HeLa cells .

How should I analyze ETV5 expression data in the context of cancer research?

When analyzing ETV5 expression data in cancer research:

• Establish baseline expression: Compare your results with published data showing that ETV5 is abnormally highly expressed in 25 types of tumor tissues compared to adjacent normal tissues .

• Correlate with clinical parameters: ETV5 expression has been associated with unfavorable prognosis in multiple malignancies and correlates with tumor pathological stage in hepatocellular carcinoma .

• Consider hypomethylation: The ETV5 gene promoter shows hypomethylation in multiple malignancies, which may explain its overexpression. Compare methylation and expression data when available .

• Analyze pathway interactions: ETV5-related genes are enriched in tumorigenesis biological processes and signaling pathways. Consider pathway analysis tools to interpret expression data .

• Evaluate therapeutic implications: ETV5 expression is associated with differential drug sensitivity. Analyze expression data in the context of treatment response .

When using flow cytometry or immunofluorescence quantification with FITC-conjugated antibodies, establish clear positive thresholds based on control cells with known ETV5 expression levels and consider reporting both percentage of positive cells and mean fluorescence intensity.

How can I validate the specificity of my FITC-conjugated ETV5 antibody results?

To validate the specificity of FITC-conjugated ETV5 antibody results:

• Genetic validation:

  • Test antibody in ETV5 knockout or knockdown models

  • Compare staining patterns in cells with artificial ETV5 overexpression

  • Use ETV4/5 double knockout models to account for potential cross-reactivity, as these proteins show functional redundancy

• Peptide competition assays:

  • Pre-incubate antibody with excess ETV5 immunizing peptide

  • Compare results with and without peptide competition

  • Specific signal should be significantly reduced after peptide competition

• Cross-validation with different detection methods:

  • Compare results with unconjugated ETV5 antibody and fluorophore-conjugated secondary antibody

  • Validate with orthogonal methods like Western blot or RT-PCR

  • Use antibodies against different ETV5 epitopes and compare localization patterns

• Cell type specificity:

  • Test in cell types with known differential expression of ETV5

  • Compare with published subcellular localization data showing primary nuclear localization

• Control experiments:

  • Include isotype control antibodies with FITC conjugation

  • Test specificity in tissues known to express minimal ETV5

For cancer research applications specifically, validate findings using the correct cell type context, as ETV5 expression patterns and functions may vary significantly between cancer types .

How can FITC-conjugated ETV5 antibodies help investigate the role of ETV5 in embryonic stem cell differentiation?

FITC-conjugated ETV5 antibodies offer valuable insights into stem cell differentiation through:

• Temporal expression analysis: Track ETV5 expression during differentiation using:

  • Flow cytometry to quantify expression changes at population level

  • Time-lapse live-cell imaging to monitor dynamics in real-time

  • Fixed-cell immunofluorescence at critical differentiation timepoints

• Co-localization with pluripotency factors: Research shows ETV5 expression is regulated by OCT3/4 in embryonic stem cells . FITC-conjugated ETV5 antibodies enable co-localization studies with OCT3/4 and other pluripotency factors.

• Target gene analysis: ETV5 regulates stem cell-related genes including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c . Combine FITC-conjugated ETV5 antibodies with RNA FISH probes to correlate ETV5 protein levels with target gene expression.

• Differentiation pathway monitoring: ETV4/5 double knockout ES cells show impaired expression of ectoderm marker genes (Fgf5, Sox1, Pax3) . Use FITC-conjugated ETV5 antibodies with antibodies against lineage markers to track differentiation patterns.

• Functional redundancy analysis: ETV4 and ETV5 show functional redundancy . FITC-conjugated antibodies enable quantitative comparison of expression patterns during differentiation.

Research indicates that artificial expression of ETV4 and/or ETV5 in Etv4/5 double knockout ES cells induces re-expression of Tcf15 and Gbx2, suggesting a mechanistic pathway for investigation using FITC-conjugated antibodies .

What approaches can be used to study the relationship between ETV5 and drug resistance mechanisms?

To investigate ETV5's role in drug resistance:

• Expression correlation with drug sensitivity: Recent studies show ETV5 expression correlates with differential drug sensitivity in multiple malignancies . FITC-conjugated antibodies can:

  • Rapidly screen patient samples for ETV5 expression

  • Sort cells based on ETV5 expression levels for downstream drug sensitivity assays

  • Monitor ETV5 expression changes during drug treatment

• EZH2-ETV5 axis investigation: Evidence indicates ETV5 facilitates cell proliferation and reduces sensitivity to the EZH2 inhibitor GSK126 via regulating EZH2 . Research approaches include:

  • Co-immunoprecipitation followed by Western blot

  • Proximity ligation assays using FITC-conjugated ETV5 antibodies

  • ChIP-seq to identify shared genomic targets

• Combinatorial drug screening: Test combinations of:

  • EZH2 inhibitors with other epigenetic modifiers

  • Conventional chemotherapeutics with epigenetic drugs

  • Targeted therapies against pathways regulated by ETV5

• Mechanistic studies:

  • Generate ETV5 knockout/knockdown models using CRISPR-Cas9

  • Test drug sensitivity before and after ETV5 modulation

  • Perform rescue experiments with wild-type or mutant ETV5

• Clinical correlation: Use FITC-conjugated ETV5 antibodies to:

  • Stratify patient samples based on ETV5 expression

  • Correlate expression with treatment outcomes

  • Develop potential companion diagnostics for EZH2 inhibitor therapy

The relationship between ETV5 and epigenetic modification offers promising avenues for overcoming drug resistance, particularly in cancers with abnormally high ETV5 expression .

How can multiplexed imaging approaches with FITC-conjugated ETV5 antibodies advance cancer biomarker research?

Multiplexed imaging with FITC-conjugated ETV5 antibodies enables sophisticated cancer biomarker research:

• Spatial heterogeneity analysis:

  • Map ETV5 expression across tumor microenvironments

  • Correlate with immune cell infiltration markers

  • Identify distinct tumor regions with differential ETV5 expression

• Multi-marker prognostic panels:

  • Combine ETV5 detection with established biomarkers

  • Create multiparameter prognostic signatures

  • Develop quantitative scoring systems incorporating spatial information

• Pathway activation mapping:

  • Simultaneously detect ETV5 with downstream targets

  • Visualize signaling pathway activation states

  • Correlate with cell proliferation and apoptosis markers

• Tumor-stroma interaction studies:

  • Examine ETV5 expression at tumor-stroma interfaces

  • Correlate with extracellular matrix components

  • Investigate relationship with invasion markers

• Technology platforms:

  • Conventional multiplexed immunofluorescence (3-5 markers)

  • Cyclic immunofluorescence (CycIF) for 20+ markers on the same sample

  • Mass cytometry imaging (MIBI, IMC) for 40+ markers with FITC-conjugated ETV5 as reference