ETC3 Antibody

What is ETV3 and what is its biological function?

ETV3 (ETS variant transcription factor 3) functions as a transcriptional repressor that contributes to growth arrest during terminal macrophage differentiation by repressing target genes involved in Ras-dependent proliferation. It belongs to the ETS protein family and represses MMP1 promoter activity . In humans, the canonical protein has 512 amino acid residues with a mass of approximately 57 kDa and is primarily localized in the nucleus . ETV3 is also known by several alternative names including PE-1, PE1, METS, ETS domain transcriptional repressor PE1, and Mitogenic Ets transcriptional suppressor . Gene orthologs have been identified in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken .

What applications can ETV3 antibodies be used for?

ETV3 antibodies are primarily used for the immunodetection of the protein encoded by the ETV3 gene. Based on available research data, validated applications include:

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immunofluorescence (IF) and Immunocytochemistry (ICC)

• Enzyme-linked immunosorbent assay (ELISA)

• Flow cytometry (FCM) for selected antibody clones

• Dot blot (DB) for certain antibody formulations

What types of ETV3 antibodies are available for research?

Several types of ETV3 antibodies are commercially available:

How should I optimize Western blot protocols for ETV3 detection?

Based on published data, the following optimization parameters are recommended for ETV3 Western blot:

• Antibody concentration: 0.1 μg/mL has been successfully used with antibody ab176717

• Sample loading: 15-50 μg of total protein from whole cell lysates

• Cell lines with reliable expression: Jurkat, HeLa, and 293T cells show detectable ETV3 expression

• Detection method: ECL technique with approximately 3-minute exposure time

• Expected band size: 57 kDa
  For optimal results:

• Include positive controls using cell lines known to express ETV3

• Consider using gradient gels (8-12%) to achieve optimal separation around the 57 kDa region

• For challenging samples, immunoprecipitation prior to Western blot may enhance detection sensitivity

What are the best practices for immunoprecipitation of ETV3?

Successful immunoprecipitation of ETV3 has been reported using the following approach:

• Starting material: 1 mg of whole cell lysate (Jurkat cells have been used successfully)

• Antibody concentration: 6 μg antibody per mg of lysate

• Loading for subsequent analysis: 20% of immunoprecipitated material
  To improve IP efficiency:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Optimize antibody incubation time and temperature (typically 2-4 hours at 4°C or overnight)

• Adjust wash stringency based on antibody specificity and background levels

How can I use ETV3 antibodies to study macrophage differentiation?

Since ETV3 contributes to growth arrest during terminal macrophage differentiation by repressing target genes involved in Ras-dependent proliferation , antibodies against ETV3 can be valuable tools in studying this process:
Experimental approach:

• Monitor ETV3 expression levels during different stages of macrophage differentiation using Western blot

• Use immunofluorescence to track subcellular localization changes during differentiation

• Combine with ChIP (Chromatin Immunoprecipitation) to identify ETV3 target genes during differentiation

• Correlate ETV3 expression with cellular proliferation markers
  Cell models:

• Primary monocytes induced to differentiate into macrophages

• Monocytic cell lines treated with differentiation-inducing agents

• Bone marrow-derived macrophages at various stages of maturation

What approaches can be used to detect different ETV3 isoforms?

ETV3 is reported to have 2 different isoforms due to alternative splicing . To differentiate between these isoforms:
Western blot approach:

• Use higher resolution gel systems (e.g., 10-12% gels with longer run times)

• Consider using antibodies targeting different epitopes that may be present/absent in specific isoforms

• Include isoform-specific positive controls if available
  PCR-based validation:

• Design primers spanning splice junctions to specifically amplify each isoform

• Quantify isoform expression using qRT-PCR with isoform-specific primers

• Validate PCR results with Western blot to confirm protein expression

How do different ETV3 antibody clones compare in specificity and application versatility?

Different ETV3 antibody clones exhibit varying specificities and application performance:

How do I troubleshoot common issues with ETV3 antibody experiments?

Common issues and troubleshooting approaches for ETV3 antibody experiments:
Weak or no signal in Western blot:

• Increase antibody concentration (try 0.2-0.5 μg/mL if 0.1 μg/mL is insufficient)

• Extend primary antibody incubation time (overnight at 4°C)

• Ensure target protein is expressed in your sample (use Jurkat cells as positive control)

• Try different detection systems (e.g., more sensitive chemiluminescent substrates)
  High background:

• Increase blocking time or concentration

• Use more stringent washing conditions

• Titrate primary antibody to optimal concentration

• Try different blocking agents (BSA vs. milk)
  Multiple bands:

• Verify if bands represent isoforms, degradation products, or non-specific binding

• Include appropriate controls (lysates from cells with known ETV3 expression)

• Increase gel resolution to better separate closely migrating bands

How can I validate the specificity of my ETV3 antibody?

Validating antibody specificity is crucial for generating reliable research data:
Experimental approaches for validation:

• Western blot with positive control lysates (e.g., Jurkat cells) should show a band at the expected 57 kDa size

• Peptide competition assays using the immunizing peptide

• Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout of ETV3 to confirm signal specificity

• Compare multiple antibodies recognizing different epitopes of ETV3

• Consider orthogonal detection methods to confirm protein identification

Can ETV3 antibodies be incorporated into antibody-epitope conjugates (AECs)?

Antibody-epitope conjugates represent promising modalities for immunotherapy applications. Based on recent research, incorporating ETV3 antibodies into AEC systems would require consideration of several conjugation strategies:
Conjugation methods for AEC development:

• Chemical conjugation: Thiol-maleimide reaction to reduced cysteine side chains

• Enzymatic conjugation: Heavy chain C-terminal conjugation using sortase A

• Genetic fusion: Direct fusion to the heavy chain C-terminus
  The epitope-to-antibody ratio (EAR) can be estimated or determined by Hydrophobic Interaction Chromatography (HIC), SDS-PAGE, or intact liquid chromatography-mass spectrometry (LC-MS) .

How can artificial intelligence approaches aid in developing improved ETV3 antibodies?

Recent advances in AI for antibody engineering offer promising approaches for developing enhanced ETV3 antibodies:
AI-based antibody design methods:

• Pre-trained Antibody generative Large Language Models can generate de novo artificial antibodies with desired binding properties

• Binding prediction models can pair antigen epitope sequences with antibody sequences to predict binding specificity and affinity

• Multi-objective linear programming with diversity constraints can create diverse and high-performing antibody libraries
  As demonstrated with other antibodies, these approaches can be used to:

• Design optimized complementarity-determining regions (CDRs)

• Screen mutations that might improve binding affinity

• Generate diverse antibody libraries targeting ETV3

What considerations are important when developing ETV3 antibody drug conjugates (ADCs)?

While specific ETV3-targeted ADCs aren't currently described in literature, general principles for developing ADCs would apply:
Design considerations for ADC development:

• Site-specific conjugation technologies enable production of homogeneous ADCs with well-characterized drug-to-antibody ratios (typically 2 or 4)

• Linker selection impacts stability and drug release properties (cleavable vs. non-cleavable)

• Payload potency and mechanism should match the biological context

• Design of Experiments (DOE) approach can optimize process conditions to meet key quality attributes
  Third-generation ADCs incorporate fully humanized antibodies, more potent payloads, and hydrophilic linker modulation (such as PEGylation) to improve stability and reduce off-target effects .