EVI2A Antibody

What is EVI2A and what is its cellular function?

EVI2A (ecotropic viral integration site 2A) is a membrane-localized protein with a length of 236 amino acid residues and a molecular weight of approximately 26.2 kDa in humans. Functionally, EVI2A is believed to form complexes either with itself or with other proteins within the cellular membrane, serving as a component of a cell-surface receptor system. The protein undergoes post-translational modifications, particularly glycosylation, which may be critical for its proper functioning . Current research suggests its involvement in cellular signaling pathways, though the complete spectrum of its biological activities remains under investigation .

What experimental applications are EVI2A antibodies commonly used for?

EVI2A antibodies are utilized across several experimental platforms in molecular and cellular biology research:

The selection of application should be guided by experimental questions and available validation data for specific antibody products .

What types of EVI2A antibodies are available for research use?

Researchers can access several categories of EVI2A antibodies, each with distinct properties:

Additionally, antibodies are available with reactivity to EVI2A from various species including human, mouse, rat, cow, horse, pig, bat, and monkey, allowing for comparative studies across model organisms .

How does EVI2A expression correlate with pathological conditions, particularly in cancer research?

Expression correlation data from studies examining tumor microenvironment interactions show:

Methodologically, researchers investigating EVI2A in pathological contexts should consider combining transcriptomic analysis (RNA-seq) with protein-level studies (IHC, Western blot) to establish consistent patterns across biological scales .

What mechanisms regulate EVI2A expression in normal and disease states?

Research indicates that EVI2A expression is regulated through multiple mechanisms, with epigenetic modification playing a particularly important role. Current evidence identifies EVI2A as a methylation-related gene in KIRC and other cancers, suggesting that DNA methylation status significantly impacts its transcriptional activity .

For researchers investigating regulatory mechanisms, the following methodological approaches are recommended:

• Methylation-specific PCR and bisulfite sequencing to assess promoter methylation status

• Chromatin immunoprecipitation (ChIP) to identify transcription factor binding

• Reporter gene assays to evaluate promoter activity under different conditions

• Treatment with epigenetic modifiers (e.g., 5-azacytidine) to experimentally manipulate methylation status

Additionally, researchers should consider examining microRNA regulation and histone modifications as complementary regulatory mechanisms that may interact with DNA methylation to fine-tune EVI2A expression in different cellular contexts .

How do EVI2A antibodies perform in detecting post-translational modifications?

Detection of post-translational modifications (PTMs) of EVI2A presents special challenges due to the protein's membrane localization and glycosylation status. Current evidence indicates that glycosylation is a significant PTM for EVI2A , potentially affecting antibody recognition and experimental outcomes.

For optimal detection of glycosylated EVI2A:

Researchers should select antibodies specifically validated for detecting the protein regardless of glycosylation state or choose epitopes known to be unaffected by this modification .

What are the optimal conditions for Western blot detection of EVI2A?

Western blotting for EVI2A requires specific optimization due to its membrane localization and post-translational modifications. Recommended protocol adjustments include:

• Sample preparation:

  • Use membrane protein extraction buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40)

  • Include protease inhibitors and maintain samples at 4°C throughout processing

  • Consider using specialized glycoprotein solubilization buffers if targeting glycosylated forms

• Gel electrophoresis parameters:

  • 10-12% polyacrylamide gels provide optimal resolution for the 26.2 kDa protein

  • Expected migration pattern: 26-30 kDa (unmodified) or 35-45 kDa (glycosylated forms)

  • Include positive control lysates from tissues with known EVI2A expression

• Transfer and detection:

  • Semi-dry transfer systems at 15-20V for 30-45 minutes typically yield efficient transfer

  • Block with 5% non-fat milk or BSA in TBST

  • Primary antibody incubation: overnight at 4°C at dilutions between 1:500-1:2000

  • HRP-conjugated secondary antibodies at 1:5000-1:10000 for 1 hour at room temperature

These experimental conditions should be further optimized based on specific antibody characteristics and sample types .

How should researchers design immunoprecipitation experiments for EVI2A interaction studies?

Immunoprecipitation (IP) of EVI2A presents unique challenges due to its membrane localization. The following methodological approach is recommended:

• Cell lysis optimization:

  • Use NP-40 or digitonin-based lysis buffers (0.5-1%) to solubilize membrane proteins while preserving protein-protein interactions

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Perform lysis at 4°C with gentle agitation for 30-60 minutes

• Antibody selection considerations:

  • Choose antibodies raised against regions not involved in protein-protein interactions

  • Validate antibody efficiency in preliminary Western blot experiments

  • For co-IP studies, epitope-tagged EVI2A constructs may provide higher specificity

• Protocol optimization:

  • Pre-clear lysates with Protein A/G beads for 1 hour at 4°C

  • Use 2-5 μg antibody per 500 μg of total protein

  • Extend incubation time to overnight at 4°C with gentle rotation

  • Include appropriate negative controls (isotype control antibodies)

• Analysis of interacting partners:

  • Mass spectrometry-based identification of co-precipitated proteins

  • Validation of key interactions through reciprocal co-IP and proximity ligation assays

This approach maximizes the likelihood of preserving physiologically relevant interactions while minimizing non-specific binding .

What controls are essential when using EVI2A antibodies in research applications?

Rigorous experimental design for EVI2A antibody applications should include several critical controls:

For immunohistochemistry or immunofluorescence, additional tissue-specific controls and counterstaining should be employed to verify subcellular localization patterns .

How can researchers address non-specific binding issues with EVI2A antibodies?

Non-specific binding is a common challenge when working with membrane protein antibodies. For EVI2A antibodies, consider these methodological solutions:

• Antibody selection refinement:

  • Choose monoclonal antibodies for higher specificity when possible

  • Review validation data showing specificity across multiple applications

  • Select antibodies targeting unique regions of EVI2A with minimal homology to related proteins

• Protocol optimization:

  • Increase blocking stringency (5% BSA or 5-10% normal serum from secondary antibody host species)

  • Add 0.1-0.3% Triton X-100 to blocking and antibody dilution buffers

  • Extend blocking time to 2 hours at room temperature

  • Perform additional washing steps (5-6 washes of 10 minutes each)

  • Titrate primary antibody concentration through dilution series experiments

• Sample preparation improvements:

  • Pre-absorb antibodies with proteins from species of non-interest

  • Use freshly prepared samples to reduce degradation products

  • Consider antigen retrieval optimization for fixed samples

• Data interpretation:

  • Compare multiple antibodies targeting different EVI2A epitopes

  • Correlate protein detection with mRNA expression data

  • Use genetic manipulation (siRNA, CRISPR) to validate specificity

These approaches should significantly reduce non-specific binding while preserving authentic EVI2A signal .

What are the challenges in detecting low abundance EVI2A in certain cell types?

EVI2A detection may be challenging in cell types with naturally low expression levels. Implementation of signal amplification strategies can help overcome this limitation:

• Enhanced protein extraction:

  • Scale up starting material (3-5x typical amounts)

  • Implement subcellular fractionation to concentrate membrane proteins

  • Use specialized extraction buffers optimized for glycosylated membrane proteins

• Signal amplification techniques:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Use high-sensitivity ECL substrates for Western blotting

  • Consider biotin-streptavidin amplification systems

  • Implement polymeric detection systems for IHC

• Sample enrichment prior to analysis:

  • Perform immunoprecipitation before Western blotting

  • Use lectin-based enrichment for glycosylated forms

  • Consider using more sensitive detection methods like droplet digital PCR at the mRNA level to correlate with protein findings

• Innovative detection platforms:

  • Single molecule array (Simoa) technology for ultra-sensitive protein detection

  • Proximity extension assay (PEA) for detecting EVI2A in complex samples

  • Mass cytometry (CyTOF) for simultaneous detection of multiple parameters

These approaches can lower detection limits by 10-100 fold compared to standard methods .

How do different sample preparation methods affect EVI2A antibody performance?

Sample preparation significantly impacts the detection of membrane-associated proteins like EVI2A. The following methodological comparison may guide researchers:

For optimal results, researchers should test multiple preparation methods when establishing a new experimental system, particularly when working with novel antibodies or tissue sources .

How can EVI2A antibodies contribute to cancer biomarker research?

EVI2A antibodies serve as valuable tools in cancer biomarker research, particularly given the protein's emerging role in multiple malignancies. Methodological approaches for biomarker application include:

• Tissue microarray analysis:

  • Quantitative immunohistochemistry across tumor stages and grades

  • Correlation with patient outcomes using appropriate statistical methods

  • Multivariate analysis incorporating other established biomarkers

• Liquid biopsy development:

  • Detection of EVI2A in circulation using highly sensitive immunoassays

  • Analysis of EVI2A in extracellular vesicles from patient serum

  • Correlation of EVI2A levels with disease progression

• Multiparameter analysis:

  • Integration with other biomarkers in predictive models

  • Development of EVI2A-based prognostic panels

  • Combined analysis with genetic and epigenetic markers

Recent research specifically demonstrates EVI2A's significant diagnostic value in kidney renal clear cell carcinoma (KIRC) with an impressive AUC of 0.906, indicating excellent discriminatory power. Furthermore, its expression correlates with tumor stage, grade, and patient survival, suggesting potential utility in stratifying patients for treatment selection and monitoring disease progression .

What is the role of EVI2A in immune cell function and how can antibodies help elucidate this?

EVI2A exhibits significant immunological associations that researchers can investigate using specialized antibody-based approaches:

• Immune cell expression profiling:

  • Multicolor flow cytometry to map EVI2A expression across immune cell subsets

  • Single-cell analyses to identify specific populations with differential expression

  • Sequential immunophenotyping during immune cell activation/differentiation

• Functional studies in immune contexts:

  • Investigation of EVI2A in immune synapse formation using high-resolution microscopy

  • Analysis of signaling pathway activation in EVI2A-expressing immune cells

  • Correlation with immune activation markers in different pathological states

• Tumor microenvironment investigations:

  • Dual immunofluorescence for EVI2A and immune cell markers in tumor sections

  • Analysis of EVI2A expression in tumor-infiltrating lymphocytes

  • Correlation with immune checkpoint molecule expression

Current research indicates significant associations between EVI2A expression and specific immune cell populations, including T follicular helper cells, CD4+ memory T cells, and CD8+ T cells. Additionally, EVI2A expression correlates with tumor microenvironment scores and may indicate increased sensitivity to immune checkpoint inhibitors targeting PD-1/CTLA-4, suggesting potential implications for immunotherapy response prediction .

What emerging technologies are enhancing the utility of EVI2A antibodies in research?

Advanced technological platforms are expanding the applications of EVI2A antibodies in research settings:

• Single-cell technologies:

  • Mass cytometry (CyTOF) for high-dimensional protein profiling

  • Single-cell proteomics with antibody-based detection

  • Spatial transcriptomics combined with protein detection for tissue mapping

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Expansion microscopy for enhanced visualization of subcellular structures

  • Live-cell imaging with antibody fragments for dynamic studies

• Antibody engineering innovations:

  • Development of single-domain antibodies (nanobodies) for enhanced penetration and reduced size

  • BiTE (Bispecific T-cell Engager) antibodies for functional studies

  • Computationally designed antibodies with enhanced specificity and affinity

• Therapeutic development platforms:

  • Antibody-drug conjugates targeting EVI2A in cancer models

  • CAR-T approaches utilizing EVI2A as a target in appropriate malignancies

  • Development of therapeutic antibodies against EVI2A for targeted therapy

Recent advances in computational antibody design, as demonstrated by the fine-tuned RFdiffusion network capable of designing de novo antibody variable heavy chains (VHH's), may soon allow for custom-designed anti-EVI2A antibodies with precisely engineered binding properties for specific research and therapeutic applications .