ETV5 Antibody, HRP conjugated

What is the biological significance of ETV5 in cancer research?

ETV5 functions as a transcription factor that contributes significantly to tumor growth and progression, particularly in colorectal cancer (CRC). Gene Ontology analysis of RNA-seq data has revealed that ETV5 plays a crucial role in regulating the cell cycle . In vitro and in vivo experiments have confirmed that ETV5 upregulation enhances tumor proliferative capacity and promotes the transition from G1 phase to S phase in the cell cycle . This transcription factor binds to specific DNA sequences containing the consensus nucleotide core sequence 5'-GGAA-3', allowing it to regulate gene expression .

Mechanistically, ETV5 has been shown to transcriptionally inhibit p21 expression, which normally functions as a cell cycle inhibitor by binding to cyclin-dependent kinases (CDKs) . When ETV5 is highly expressed, p21 levels decrease, resulting in increased CDK activity, enhanced phosphorylation of proteins like p130 (a member of the RB family), and accelerated G1/S transition . Importantly, clinical data has demonstrated that CRC patients with high ETV5 expression and low p21 expression show the worst prognosis, highlighting ETV5's potential as both a diagnostic and prognostic marker .

What experimental applications are optimal for HRP-conjugated ETV5 antibodies?

HRP-conjugated ETV5 antibodies are versatile tools for multiple experimental applications in cancer research and molecular biology. Western blotting represents a primary application, with ETV5 typically detected at approximately 70 kDa, though the calculated molecular weight is 57.838 kDa . For optimal results in Western blot applications, membranes should be blocked in 3% milk in TBS-T (0.1% Tween®) before overnight incubation with the antibody at 4°C at a dilution of approximately 1:500 .

Immunocytochemistry/immunofluorescence (ICC/IF) represents another valuable application for visualizing ETV5 cellular localization. Protocols typically involve fixing cells with 4% paraformaldehyde at room temperature for 15 minutes, followed by incubation with the primary antibody at a 1:500 dilution . Additional applications include ELISA and flow cytometry, which are particularly useful for quantitative assessments of ETV5 expression levels in cell populations .

For chromatin immunoprecipitation (ChIP) experiments investigating ETV5's role as a transcription factor, HRP-conjugated antibodies can help identify specific binding sites in promoter regions, such as the documented binding of ETV5 to sites in the p21 promoter . The enzyme conjugation provides enhanced sensitivity for detection in these various applications, making HRP-conjugated ETV5 antibodies particularly valuable for examining endogenous expression levels.

What are the critical experimental controls needed when working with ETV5 antibodies?

When designing experiments with ETV5 antibodies, several critical controls must be included to ensure valid and interpretable results. For Western blot applications, loading controls such as GAPDH antibodies must be incorporated to normalize protein levels across samples . Including positive controls from cell lines with known ETV5 expression is essential—HCT116 cells have been documented to have low ETV5 expression levels, while RKO cells demonstrate high expression levels .

Negative controls should involve knockdown experiments using ETV5-specific shRNA or siRNA to confirm antibody specificity . The research shows that knocking down ETV5 in RKO cells significantly reduced its expression as verified by RT-qPCR and Western blotting, providing a reliable negative control system . For immunohistochemistry or immunofluorescence applications, secondary antibody-only controls are necessary to assess non-specific binding.

For functional studies examining ETV5's role in cell cycle regulation, flow cytometry analysis following ETV5 manipulation provides essential control data. Studies have demonstrated that ETV5 overexpression significantly decreased the proportion of cells in G1 phase while increasing the proportion in S phase, with knockdown experiments showing consistent reciprocal results . When investigating downstream targets like p21, parallel experiments manipulating both ETV5 and target gene expression provide crucial mechanistic controls—p21 knockdown using siRNA has been shown to significantly increase cell proliferation ability and promote G0/G1 to S phase transition .

What are the optimal storage and handling conditions for maintaining ETV5 antibody activity?

Proper storage and handling of ETV5 antibodies is critical for maintaining their specificity and activity over time. For long-term storage, lyophilized antibody preparations should be kept at -20°C for up to one year from the date of receipt . The lyophilized form typically contains stabilizing components such as trehalose, NaCl, Na₂HPO₄, and may include NaN₃ as a preservative .

For reconstitution, adding 0.2ml of distilled water to lyophilized product typically yields a usable concentration of approximately 500μg/ml . After reconstitution, the antibody can be stored at 4°C for approximately one month for regular use . For longer storage periods after reconstitution, it's advisable to create smaller aliquots and store them at -20°C for up to six months . This aliquoting process is crucial as it minimizes exposure to repeated freeze-thaw cycles, which can significantly degrade antibody performance.

When handling the antibody during experimental procedures, maintain cold chain principles—keep the antibody on ice when in use and return to appropriate storage temperatures promptly after use. For diluted working solutions used in applications such as Western blotting or immunofluorescence, prepare fresh dilutions when possible or store at 4°C for no more than 24-48 hours to preserve optimal activity and minimize background signal.

How can ChIP-seq with ETV5 antibodies be optimized to identify novel transcriptional targets?

Optimizing ChIP-seq with ETV5 antibodies requires careful consideration of several experimental parameters to ensure high-quality data generation. Based on previous research findings, ETV5 binds to specific sites in target gene promoters, such as the documented binding to sites in the p21 promoter region . When designing ChIP-seq experiments, prioritize surveying sequences upstream of potential target genes, typically ranging from -2000bp to 0bp with respect to the transcription start site where ETV5 binding sites have been previously identified .

For chromatin preparation, aim for DNA fragments between 200-300bp to achieve optimal resolution for identifying precise binding sites. The sonication protocol should be carefully optimized for each cell type used, with pilot experiments to confirm proper fragmentation. Regarding antibody usage, pre-clearing chromatin with protein A/G beads helps reduce background signal, while antibody concentrations should be empirically determined—typically starting with 3-5μg per reaction for transcription factor ChIP.

After identifying potential binding sites through ChIP-seq analysis, validation through complementary methods is essential. The research demonstrates that luciferase reporter assays effectively confirm functional significance of binding sites, as shown when mutation of site A in the p21 promoter abolished ETV5-mediated inhibition . For computational analysis, focus on identifying the known consensus ETV5 binding motif (5'-GGAA-3') while also searching for extended motifs that might confer additional specificity.

Integration of ChIP-seq data with RNA-seq following ETV5 manipulation (overexpression or knockdown) provides powerful insights into the functional consequences of binding. This approach successfully identified the regulatory relationship between ETV5 and p21, revealing that ETV5 binds to the p21 promoter and represses its expression in a p53-independent manner .

How should researchers interpret complex cell cycle effects when manipulating ETV5 expression?

Interpreting the complex effects of ETV5 manipulation on cell cycle progression requires multi-dimensional analysis and careful experimental design. Flow cytometry analysis represents a fundamental approach for quantifying cell cycle distribution changes, as demonstrated in research where ETV5 overexpression significantly decreased the proportion of cells in G1 phase while increasing S phase populations . When knockdown experiments were performed in RKO cells, researchers observed the inverse effect, confirming ETV5's specific role in promoting G1/S transition .

Beyond simple cell cycle distribution analysis, researchers should examine the molecular mechanisms underlying these changes. The p21-CDK pathway has been identified as a critical mediator of ETV5's cell cycle effects . After identifying p21 as a downstream target of ETV5 through PCR array analysis, researchers confirmed that p21 knockdown resulted in faster cancer cell growth and more cells transitioning from G0/G1 into S phase . Co-immunoprecipitation experiments further revealed that p21 binds to CDK2, CDK4, and CDK6, inhibiting p130 phosphorylation—a critical step in cell cycle progression .

When designing experiments to interpret these complex effects, researchers should incorporate multiple readouts beyond cell cycle distribution. These include proliferation assays (such as the CCK-8 assay and colony formation assays used in the research), analysis of CDK substrate phosphorylation (particularly p130), and expression analysis of multiple cell cycle regulators . In vivo validation provides additional crucial context—studies showed that ETV5 overexpression resulted in larger tumors with increased expression of proliferation markers Ki67 and PCNA .

For comprehensive interpretation, researchers should also investigate how ETV5's cell cycle effects influence therapeutic responses. The research demonstrated that by targeting p21 to regulate CDK function, ETV5 altered cancer cell sensitivity to CDK inhibitors such as palbociclib and dinaciclib, highlighting the clinical relevance of these mechanistic findings .

How can researchers effectively use ETV5 antibodies in multiplex immunofluorescence studies?

Implementing multiplex immunofluorescence with ETV5 antibodies requires strategic planning to overcome technical challenges while generating meaningful co-localization data. When designing these experiments, antibody selection represents a critical first step—choose primary antibodies from different host species to avoid cross-reactivity issues, or if multiple antibodies from the same species are necessary, employ sequential staining protocols with complete stripping between rounds .

For sample preparation, paraformaldehyde fixation (4% for 15 minutes at room temperature) has been successfully used for ETV5 detection in cells, establishing a baseline protocol for immunofluorescence applications . Permeabilization conditions must be carefully optimized, especially when analyzing nuclear proteins like ETV5, which requires complete nuclear permeabilization while preserving epitope integrity.

When establishing staining protocols, primary antibody dilutions typically range from 1:100 to 1:500 for ETV5 detection in immunofluorescence applications . For multiplex protocols examining ETV5 alongside cell cycle regulators like p21, parallel staining of sequential sections provides valuable comparative data while avoiding potential cross-reactivity issues. High-resolution confocal microscopy with appropriate filter sets is essential for clearly distinguishing between fluorophores and accurately assessing co-localization.

Quantitative analysis of co-localization requires specialized software implementing established algorithms such as Pearson's or Mander's correlation coefficients. When studying ETV5's relationship with other nuclear proteins, especially transcription factors, be mindful that co-localization may occur at specific nuclear foci rather than throughout the entire nucleus, necessitating sub-nuclear resolution imaging and analysis. For functional interpretation, correlate multiplex immunofluorescence findings with mechanistic data, such as the demonstrated negative correlation between ETV5 and p21 expression in colorectal cancer tissues .

What are effective strategies for combining ETV5 antibody detection with functional CDK inhibitor studies?

Integrating ETV5 antibody detection with CDK inhibitor studies requires methodical experimental design to elucidate the relationship between ETV5 expression and drug sensitivity. Research has demonstrated that ETV5 targeting inhibits p21 expression, thereby regulating CDK function and altering sensitivity to CDK inhibitors like palbociclib and dinaciclib . To effectively combine these approaches, researchers should first establish baseline ETV5 expression in their experimental models using Western blotting or immunofluorescence with validated antibodies .

For controlled manipulation of ETV5 levels, implement parallel experiments with ETV5 overexpression and knockdown systems. Research showed that cell viability increased when ETV5 was overexpressed in the HCT116 cell line treated with palbociclib (a selective CDK4/6 inhibitor), while cell viability was reduced after knockdown of ETV5 in the RKO cell line—with dinaciclib (another CDK inhibitor) showing consistent results .

When designing drug response assays, incorporate dose-response curves rather than single-point measurements to fully characterize how ETV5 levels affect the potency (EC50) and efficacy (maximum effect) of CDK inhibitors. Monitor not only viability but also more specific readouts of CDK inhibition, such as phosphorylation status of CDK substrates. The research demonstrated that ETV5 promotes phosphorylation of p130 (an RB family protein), with this phosphorylation being reduced after ETV5 knockdown .

How can researchers address non-specific binding issues with ETV5 antibodies in complex tissue samples?

Non-specific binding represents a significant challenge when using ETV5 antibodies in complex tissue samples, requiring systematic optimization to achieve specific detection. For tissue preparation, optimize fixation protocols—excessive fixation can mask epitopes while insufficient fixation may compromise tissue morphology. The research used formalin-fixed, paraffin-embedded tissues for ETV5 detection, establishing a baseline protocol for immunohistochemical analysis .

Blocking procedures require careful optimization—extend blocking times (60 minutes minimum) and test different blocking agents beyond the standard 3% milk used in Western applications . For tissue immunohistochemistry, 5-10% normal serum from the same species as the secondary antibody often provides superior blocking. Sample-specific autofluorescence (particularly in tissues with high collagen content) can be reduced using specialized quenching protocols prior to antibody incubation.

Antibody dilution optimization is crucial—while a 1:500 dilution has been reported for cell-based immunofluorescence , tissue applications typically require more concentrated antibody solutions. Implement a systematic titration experiment testing multiple concentrations to determine the optimal signal-to-noise ratio for your specific tissue type. When background persists despite optimization, consider employing biotinylated secondary antibodies with streptavidin-HRP detection systems, which can provide enhanced specificity.

Validation controls are essential for distinguishing specific from non-specific signals. The research demonstrated a clear negative correlation between ETV5 and p21 expression in colorectal cancer tissues , providing a built-in biological control—areas with high ETV5 should show correspondingly low p21 staining. For technical validation, include isotype controls and blocking peptide competition controls where the antibody is pre-incubated with the immunizing peptide to confirm signal specificity .

What approaches can overcome challenges in detecting low abundance ETV5 in clinical samples?

Detecting low abundance ETV5 in clinical samples presents technical challenges that require specialized approaches to enhance sensitivity. Signal amplification techniques represent a primary strategy—implement tyramide signal amplification (TSA) for HRP-conjugated antibodies, which can increase detection sensitivity by 10-100 fold compared to conventional methods. For immunohistochemical applications, extended chromogen development times with careful monitoring can enhance detection of low expression levels.

Sample enrichment approaches can significantly improve detection capabilities. Since ETV5 is a nuclear transcription factor, employing nuclear extraction protocols before Western blotting concentrates the target protein. The research has established that different cell lines exhibit varying ETV5 expression levels, with HCT116 showing low expression and RKO showing high expression , providing important reference points for assessing detection sensitivity in clinical samples.

Protocol modifications can enhance antibody binding efficiency. Extended primary antibody incubation (overnight at 4°C instead of 1-2 hours at room temperature) allows more complete epitope binding. Reducing washing stringency slightly (using lower salt concentrations or detergent levels) can preserve weak signals, though this must be balanced against potential increases in background.

Alternative detection strategies may be necessary for extremely low abundance situations. Consider proximity ligation assay (PLA) which can detect protein interactions with higher sensitivity than conventional immunodetection. RNA-based detection methods like RNA in situ hybridization or RT-qPCR provide alternative measures of ETV5 expression when protein detection proves challenging. For advanced applications, consider laser capture microdissection to isolate specific cell populations from heterogeneous clinical samples, concentrating cells with potential ETV5 expression before analysis .

How should researchers address discrepancies between ETV5 detection in Western blot versus immunohistochemistry?

Discrepancies between Western blot and immunohistochemistry (IHC) results for ETV5 detection require systematic analysis to determine whether they represent technical artifacts or biologically meaningful differences. Methodological differences fundamentally impact detection—Western blot detects denatured ETV5 protein in whole cell/tissue lysates, while IHC visualizes the protein in its native conformation within preserved cellular architecture .

Epitope accessibility variations often underlie these discrepancies. In Western blot, complete denaturation exposes all potential epitopes, while in IHC, fixation and processing can mask certain epitopes. The commercially available ETV5 antibodies are designed against specific regions (e.g., aa 200-450 in one product, and position D72-Q246 in another) , and these regions may have differential accessibility in the two methods. When encountering discrepancies, consider testing multiple antibodies targeting different ETV5 epitopes.

Sample heterogeneity represents another critical factor, particularly for clinical specimens. IHC reveals spatial expression patterns that may be averaged in Western blot analysis of whole tissue lysates. The research demonstrated a negative correlation between ETV5 and p21 expression in colorectal cancer tissues using IHC with H-score quantification , highlighting the importance of standardized scoring methods for meaningful cross-method comparisons.

For resolution of discrepancies, implement orthogonal validation approaches. Correlate both detection methods with mRNA analysis (RT-qPCR or RNA-seq) to provide additional context. Consider biological functionality—the research established that ETV5 promotes cell proliferation and G1/S transition , so correlating expression data from both methods with functional readouts like Ki67 or PCNA staining can help determine which method better reflects the biological activity of ETV5 in your system.

What strategies can ensure reproducible quantification of ETV5 levels across different experimental platforms?

Achieving reproducible quantification of ETV5 levels across different experimental platforms requires standardized workflows and rigorous normalization strategies. For Western blot quantification, implement consistent sample preparation protocols—uniform lysis buffers, protein determination methods, and loading amounts (typically 20-30μg total protein). Include recombinant ETV5 protein standards at known concentrations to generate calibration curves for absolute quantification when necessary.

Normalize Western blot data to appropriate loading controls—the research utilized GAPDH as a loading control for ETV5 Western blot analysis . For transcription factors like ETV5 that localize primarily to the nucleus, consider using nuclear-specific loading controls like Lamin B1 for more accurate normalization. Employ image analysis software with background subtraction capabilities to quantify band intensities, and report results as normalized ratios rather than absolute values.

For immunohistochemical quantification, the research implemented the H-score system for ETV5 and p21, which combines staining intensity and percentage of positive cells . This standardized scoring approach enables more objective comparison across specimens and studies. Automated image analysis systems can further enhance objectivity and reproducibility of IHC quantification. For immunofluorescence applications, calibrate exposure settings using control samples to avoid saturation, and use identical acquisition parameters across all experimental conditions.

For cross-platform integration, establish conversion factors by analyzing the same samples across different methods. Correlation analysis between methods (e.g., plotting Western blot densitometry values against IHC H-scores for the same samples) can identify systematic differences and allow for appropriate corrections. Reference standards—cell lines with well-characterized ETV5 expression levels like RKO (high expression) and HCT116 (low expression) —should be included in each experimental batch to enable inter-experimental normalization.

How can ETV5 expression patterns guide patient stratification for targeted cell cycle therapies?

For implementing ETV5-based stratification, standardized assessment protocols are essential. Immunohistochemical analysis using the H-score system, which combines staining intensity and percentage of positive cells, provides a quantitative approach for categorizing ETV5 and p21 expression levels . A multi-marker approach integrating both ETV5 and p21 status yields more refined stratification than either marker alone. Primary stratification categories should include: High ETV5/Low p21 (highest risk), Low ETV5/High p21 (better prognosis), and intermediate patterns .

For therapeutic decision-making, the research demonstrated that ETV5 expression levels affect sensitivity to CDK inhibitors. Cell viability increased when ETV5 was overexpressed in cells treated with palbociclib (CDK4/6 inhibitor) or dinaciclib, while ETV5 knockdown reduced viability . This suggests that patients with high ETV5 expression might benefit from higher doses of CDK inhibitors or combination approaches targeting the ETV5-p21 axis alongside CDK inhibition.

Implementation requires developing standardized cutoff values for "high" versus "low" expression based on reference populations, and prospective validation studies correlating expression patterns with treatment outcomes. The mechanistic understanding that ETV5 regulates cell cycle progression through p21 inhibition and subsequent effects on CDK function and p130 phosphorylation provides a scientific rationale for this stratification approach and helps predict which therapeutic interventions might overcome ETV5-mediated cell cycle dysregulation .

What are the best practices for analyzing ETV5 in circulating tumor cells or liquid biopsies?

Analyzing ETV5 in circulating tumor cells (CTCs) or liquid biopsies requires specialized approaches that address the unique challenges of these sample types. For CTC isolation, epithelial cell adhesion molecule (EpCAM)-based capture systems may be effective for epithelial-origin cancers, but ETV5 analysis should consider potential heterogeneity in EpCAM expression. Size-based filtration methods offer an alternative approach less dependent on specific surface markers.

Once CTCs are isolated, immunofluorescence detection of ETV5 can follow protocols similar to those established for cultured cells, with paraformaldehyde fixation (4% for 15 minutes) followed by permeabilization and antibody incubation at approximately 1:500 dilution . Combined staining for ETV5 alongside epithelial markers (cytokeratins) and exclusion of leukocyte markers (CD45) helps confirm the tumor origin of analyzed cells. Since ETV5 is a transcription factor with nuclear localization, confocal microscopy with Z-stack capabilities is recommended for accurate subcellular localization assessment.

For circulating tumor DNA (ctDNA) analysis, consider examining ETV5 promoter methylation status or copy number variations as indirect measures of potential expression changes. While direct protein analysis is not possible with ctDNA, these genetic and epigenetic alterations may provide surrogate markers for altered ETV5 expression or function.

Validation and quality control are particularly critical for liquid biopsy applications. Spike-in experiments using cell lines with known ETV5 expression levels (such as RKO with high expression and HCT116 with low expression) can establish detection sensitivity limits and reproducibility. For clinical implementation, longitudinal monitoring protocols should standardize blood collection volumes, processing times, and storage conditions to minimize pre-analytical variables that could affect ETV5 detection.

How can researchers effectively validate ETV5 as a biomarker in multi-center clinical studies?

Validating ETV5 as a biomarker in multi-center clinical studies requires rigorous standardization of pre-analytical, analytical, and post-analytical processes to ensure comparable results across institutions. For specimen collection and processing, implement detailed standard operating procedures (SOPs) covering fixation methods, processing times, and storage conditions. The research successfully used formalin-fixed, paraffin-embedded tissue samples for ETV5 immunohistochemical analysis , establishing a baseline protocol for tissue-based studies.

Analytical standardization should include centralized antibody validation and lot testing to minimize reagent variability. The research demonstrates that commercially available antibodies can successfully detect ETV5 in multiple applications , but standardized protocols must specify exact antibodies, dilutions, incubation conditions, and detection systems. For immunohistochemistry, consider centralized staining or automated staining platforms to minimize inter-laboratory variations. Digital pathology with algorithm-based scoring can reduce subjective interpretation differences compared to the H-score system used in the research .

Reference standards and controls are essential for cross-center normalization. Distribute tissue microarrays containing samples with known ETV5 expression levels (including cell lines like RKO and HCT116 with established expression patterns) to all participating centers for regular quality control assessment. For quantitative assays like RT-qPCR or digital droplet PCR measuring ETV5 mRNA, include calibration standards and inter-laboratory proficiency testing.