E2-Tag Monoclonal Antibody

What is the E2-Tag and what is its biochemical composition?

The E2-Tag is a peptide sequence derived from the Bovine Papillomavirus type-1 transactivator protein E2. It consists of 10 amino acids (SSTSSDFRDR) and serves as an epitope tag for protein labeling and detection in research applications . Epitope tags like E2 are valuable molecular tools that allow researchers to track and analyze proteins of interest without interfering with their native function, particularly when specific antibodies against the protein of interest are unavailable.

The tag's relatively small size minimizes interference with protein folding or interactions while still providing a unique recognition site for highly specific antibodies. E2-Tag functions as a molecular handle that can be genetically engineered to various positions within a recombinant protein.

How do E2-Tag monoclonal antibodies function in experimental systems?

E2-Tag monoclonal antibodies specifically recognize the E2 epitope sequence when it is fused to a protein of interest. The detection mechanism relies on the binding of mouse monoclonal antibodies (such as clone 4F8 or B2-E2) that have high specificity for the E2-Tag sequence . These antibodies can recognize the E2-Tag regardless of its position within the recombinant protein (C-terminal, internal, or N-terminal), providing versatility in experimental design .

The antibodies function through standard immunological principles:

• Primary binding occurs between the anti-E2-Tag monoclonal antibody and the epitope tag on the fusion protein

• Detection systems (enzyme-conjugated secondary antibodies, fluorophores, etc.) then recognize the primary antibody

• Signal generation allows visualization or quantification of the tagged protein

This system enables researchers to detect, isolate, or visualize the tagged protein in various experimental contexts without needing antibodies against the protein itself.

What are the critical differences between various E2-Tag monoclonal antibody clones?

Different E2-Tag monoclonal antibody clones exhibit varying characteristics that can significantly impact experimental outcomes:

When selecting an E2-Tag antibody clone, researchers should consider:

• Binding affinity - higher affinity clones provide better sensitivity

• Background signal - some clones may produce lower non-specific binding

• Application compatibility - certain clones perform better in specific techniques

• Recognition efficiency - quantitative studies show some antibodies generate high signals even at low concentrations (50 ng/mL), placing them in the "good" antibody category

The selection of an appropriate clone should be guided by the specific experimental requirements and validated for each application.

What are the optimal protocols for using E2-Tag monoclonal antibodies in Western blotting?

For optimal Western blot results with E2-Tag monoclonal antibodies, follow this methodological approach:

Sample Preparation:

• Lyse cells in an appropriate buffer (RIPA buffer for most applications) containing protease inhibitors

• Quantify protein concentration (Bradford or BCA assay)

• Prepare samples in Laemmli buffer with reducing agent (β-mercaptoethanol)

• Heat samples at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Load 10-30 μg total protein per lane (adjust based on expression level)

• Separate proteins using SDS-PAGE (select gel percentage based on target protein size)

• Transfer to PVDF or nitrocellulose membrane (PVDF recommended for higher protein retention)

Immunodetection:

• Block membrane with 5% non-fat milk or 3-5% BSA in TBS-T (1 hour, room temperature)

• Incubate with anti-E2-Tag antibody at 1:5000 dilution in blocking buffer (overnight, 4°C)

• Wash 3-5 times with TBS-T (5-10 minutes each)

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000, 1 hour, room temperature)

• Wash thoroughly and develop using enhanced chemiluminescence (ECL) substrate

Controls:

• Include positive control (known E2-tagged protein)

• Include negative control (non-tagged version of protein)

• Use loading control (β-actin, GAPDH) to normalize protein amounts

This protocol typically yields specific detection of E2-tagged proteins with minimal background signal.

How can E2-Tag monoclonal antibodies be utilized in immunofluorescence applications?

E2-Tag monoclonal antibodies can be effectively employed in immunofluorescence microscopy using the following methodological approach:

Cell Preparation:

• Grow cells on glass coverslips or appropriate imaging chambers

• Transfect or transduce cells with constructs expressing E2-tagged proteins

• Fix cells using 4% paraformaldehyde (10 minutes, room temperature) for most applications

  • Alternative fixation methods (methanol, acetone) may be tested if PFA affects epitope accessibility

Immunostaining:

• Permeabilize cells with 0.1-0.3% Triton X-100 in PBS (5-10 minutes)

• Block with 3-5% BSA or 5-10% normal serum in PBS (30-60 minutes)

• Incubate with anti-E2-Tag primary antibody at 1:100-1:1000 dilution (overnight, 4°C)

• Wash 3-5 times with PBS containing 0.05% Tween-20

• Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000, 1 hour, room temperature)

• Wash thoroughly and counterstain nuclei with DAPI if desired

• Mount slides with anti-fade mounting medium

Optimization Strategies:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Test different blocking agents to minimize background

• Include detergents in antibody diluents to reduce non-specific binding

• Compare multiple fixation protocols to maximize epitope accessibility

• Consider signal amplification systems for low-abundance targets

Comparative studies have shown that high-quality E2-Tag antibodies can generate significant signals even at lower concentrations (50 ng/mL), placing them among the more efficient epitope tag detection systems .

What strategies can improve the sensitivity of E2-Tag detection in ELISA assays?

To enhance the sensitivity of E2-Tag detection in ELISA applications, implement these methodological improvements:

Antibody Selection and Preparation:

• Use high-affinity monoclonal antibodies specific to E2-Tag

• Purify antibodies to remove contaminants that may interfere with binding

• Determine optimal working concentration through titration experiments

Signal Amplification Techniques:

• Implement streptavidin-biotin detection systems (4-8× signal enhancement)

• Consider polymer-based detection systems with multiple enzyme molecules per antibody

• Utilize chemiluminescent or fluorescent substrates instead of colorimetric detection

• Extend substrate development time for increased sensitivity

Assay Format Optimization:

• Test different plate coating buffers (carbonate buffer pH 9.6 vs. PBS pH 7.4)

• Evaluate various blocking agents (BSA, casein, commercial blockers) for lowest background

• Optimize incubation times and temperatures:

  • Extended primary antibody incubation (overnight at 4°C)

  • Secondary antibody incubation (2 hours at room temperature)

• Implement sandwich ELISA format with capture and detection antibodies for maximum sensitivity

Sample Processing:

• Pre-clear samples to remove interfering substances

• Concentrate samples when target protein is in low abundance

• Optimize sample diluent composition to enhance specific binding

These optimizations can significantly improve detection limits, potentially reaching sensitivity in the low pg/mL range. Researchers should systematically test these parameters in their specific experimental context to determine the optimal conditions.

How can E2-Tag monoclonal antibodies be employed in epitope mapping studies?

E2-Tag monoclonal antibodies offer valuable tools for epitope mapping through several methodological approaches:

Fusion Protein Strategy:

• Generate a series of fusion constructs with E2-Tag positioned at various locations relative to the target epitope

• Express and purify these fusion proteins

• Perform binding assays (ELISA, Western blot) to assess accessibility of the E2-Tag

• Map regions where tag insertion disrupts antibody binding to identify critical epitope residues

Peptide Array Analysis:

• Create overlapping peptide arrays spanning the protein of interest

• Include E2-Tag as a control epitope with known antibody reactivity

• Compare binding patterns between anti-E2-Tag antibodies and antibodies against the target protein

• Identify peptides with similar binding characteristics to map epitopes

Domain Swapping:

• Engineer chimeric proteins containing domains from different proteins with E2-Tags

• Assess antibody binding to identify which domains contain the epitope of interest

• Further refine mapping by creating subdomain constructs with E2-Tags

This approach has been successfully applied in studies with viral proteins, where researchers identified linear B-cell epitopes using monoclonal antibodies . For example, with CSFV E2 protein, researchers identified specific epitopes (25GLTTTWKEYSHDLQL39 and 259GNTTVKVHASDERGP273) using similar methodologies . The same principles can be applied using E2-Tag as a control or reference point for mapping epitopes in other proteins.

What are the optimal storage conditions for E2-Tag monoclonal antibodies to maintain activity?

Proper storage of E2-Tag monoclonal antibodies is critical for maintaining their activity and specificity over time. Based on manufacturer recommendations and research practices:

Long-term Storage:

• Store concentrated antibody solutions at -20°C or -80°C

• Most commercial preparations contain 50% glycerol and 0.02% sodium azide as preservatives

• Divide antibody into small aliquots before freezing to avoid repeated freeze-thaw cycles

• Label aliquots with date, concentration, and number of freeze-thaw cycles

Working Solution Storage:

• Diluted antibody solutions can be stored at 4°C for 1-2 weeks

• Add preservatives (0.02% sodium azide) to prevent microbial growth

• For prolonged storage of working dilutions, return to -20°C

Stability Factors:

• Avoid repeated freeze-thaw cycles, which can cause protein denaturation and aggregation

• Protect from extended exposure to room temperature

• Shield from direct light, especially if conjugated to fluorophores

• Maintain sterile conditions to prevent contamination

Monitoring Antibody Activity:

• Include positive controls when using antibodies that have been stored for extended periods

• Record antibody performance over time to detect any degradation

• Consider refreshing antibody stocks if signal quality diminishes

Following these storage guidelines will help maintain antibody activity and ensure consistent experimental results across multiple studies.

How can non-specific binding be minimized when using E2-Tag antibodies?

Non-specific binding is a common challenge when using E2-Tag antibodies that can compromise experimental results. Implement these methodological approaches to minimize background:

Blocking Optimization:

• Test different blocking agents:

  • 3-5% BSA in PBS or TBS

  • 5% non-fat dry milk (not suitable for phospho-specific applications)

  • Commercial blocking solutions

• Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

• Include blocking agent in antibody diluent solutions

Antibody Preparation:

• Pre-adsorb antibodies against tissues or cells lacking the E2-Tag

• Centrifuge antibody solutions (10,000 × g, 5 minutes) before use to remove aggregates

• Titrate antibody concentration to determine the minimal effective concentration

• Use high-quality, affinity-purified antibodies

Washing Procedures:

• Increase number of washes (5-6 washes instead of standard 3)

• Extend washing time (10-15 minutes per wash)

• Add detergents to wash buffers (0.05-0.1% Tween-20 or 0.1% Triton X-100)

• Consider higher salt concentration in wash buffers (up to 500 mM NaCl) to disrupt non-specific ionic interactions

Additional Techniques:

• Add 0.1-0.3% Triton X-100 to antibody diluents to reduce hydrophobic interactions

• Include 5-10% normal serum from the species of the secondary antibody

• For Western blots, consider using PVDF membranes instead of nitrocellulose

• For immunofluorescence, include an additional permeabilization step before blocking

These optimizations should be systematically tested and adapted to specific experimental conditions, as the sources of non-specific binding can vary between applications and sample types.

What are common pitfalls in E2-Tag detection and how can they be addressed?

Researchers frequently encounter several challenges when working with E2-Tag antibodies. Here are common pitfalls and their methodological solutions:

Low Signal Intensity:

• Problem: Weak or undetectable signal despite proper expression
  Solutions:

  • Increase antibody concentration or incubation time

  • Try alternative antibody clones with higher affinity

  • Implement signal amplification systems

  • Verify tag accessibility in protein structure

  • Check if fixation methods have altered epitope conformation

High Background:
2. Problem: Excessive non-specific binding obscuring specific signal
Solutions:

• Optimize blocking conditions (agent, time, temperature)

• Increase washing stringency (more washes, longer duration)

• Reduce antibody concentration

• Pre-adsorb antibody against non-specific binding sites

• Use more specific secondary antibodies

Multiple Bands in Western Blot:
3. Problem: Detection of unexpected bands beyond target protein
Solutions:

• Verify sample integrity (add protease inhibitors)

• Optimize lysis conditions to prevent degradation

• Increase gel percentage for better resolution

• Perform peptide competition assays to identify specific bands

• Validate with alternative detection methods

Tag Inaccessibility:
4. Problem: E2-Tag is masked by protein folding
Solutions:

• Redesign construct with tag at different position (N-terminal, C-terminal)

• Add flexible linker sequences between protein and tag

• Use denaturing conditions for Western blot

• Try alternative fixation methods for immunofluorescence

• Consider mild denaturation steps before antibody incubation

Cross-Reactivity:
5. Problem: Antibody binds to endogenous proteins
Solutions:

• Include non-transfected controls

• Perform immunoprecipitation with tagged proteins

• Use siRNA to knock down target protein and confirm specificity

• Try alternative antibody clones with higher specificity

• Validate with orthogonal methods

Systematic troubleshooting using these approaches can significantly improve the reliability and specificity of E2-Tag detection in various experimental applications.

How can E2-Tag monoclonal antibodies be employed in single B-cell isolation techniques?

E2-Tag monoclonal antibodies can be utilized in sophisticated single B-cell isolation protocols, particularly for developing new monoclonal antibodies against specific targets. The methodology involves:

Protein Preparation:

• Express and purify E2-tagged target protein using appropriate expression systems

• Biotinylate the E2-tagged protein using NHS-LC-biotin reagents

• Validate biotinylation by Western blot analysis

• Quantify the degree of biotinylation to ensure optimal labeling

Immunization Strategy:

• Immunize mice with purified E2-tagged protein (typically 25 μg per dose)

• Use appropriate adjuvants (e.g., Freund's) to enhance immune response

• Administer 2-3 booster immunizations at 2-week intervals

• Validate antibody response using ELISA or other serological assays

Single B-Cell Isolation:

• Harvest splenocytes from immunized mice

• Block Fc receptors with mouse Fc block reagent

• Stain cells with:

  • Biotinylated E2-tagged protein

  • Fluorescently labeled anti-mouse IgM antibodies (e.g., IgM-FITC)

  • Anti-biotin secondary antibody (e.g., anti-biotin-APC)

• Sort E2-specific plasmablasts (E2-APC+ and IgM-FITC−) using flow cytometry

• Distribute single cells into 96-well plates containing lysis buffer

Antibody Gene Amplification:

• Perform reverse transcription using random primers

• Amplify immunoglobulin genes using nested PCR with specific primers

• Sequence VH and VL regions to identify unique antibody clones

• Clone into expression vectors for recombinant antibody production

This methodology has been successfully applied for generating monoclonal antibodies against viral proteins like CSFV E2 and can be adapted for various research targets using E2-Tag as a selection marker or reference point.

What are the considerations for using E2-Tag in structural biology applications?

When utilizing E2-Tag for structural biology studies, researchers must address several methodological considerations:

Tag Positioning Effects:

• N-terminal tagging may affect signal peptide processing or disrupt functional domains

• C-terminal tagging generally offers less interference with protein folding

• Internal tagging requires careful placement in flexible loop regions

• Consider using removable tags with protease cleavage sites for post-purification tag removal

Structural Impact Assessment:

• Compare activity of tagged vs. untagged proteins to evaluate functional consequences

• Analyze thermal stability using differential scanning fluorimetry

• Assess oligomerization state using size-exclusion chromatography

• Verify correct folding using circular dichroism spectroscopy

• Conduct limited proteolysis to identify potential structural perturbations

Crystallization Considerations:

• E2-Tag (10 amino acids) may introduce flexibility that hinders crystallization

• Include constructs with and without tags in crystallization trials

• Consider tag removal before crystallization attempts

• Use engineered crystallization chaperones as alternative to traditional tags

• Test different linker lengths between protein and tag

Cryo-EM Applications:

• E2-Tag can serve as a localization marker for subunit identification in complexes

• Position tags away from important interaction surfaces

• Tag visibility may be enhanced by fusion to larger domains for improved particle picking

• Compare structures with tags in different positions to verify structural integrity

NMR Considerations:

By systematically addressing these considerations, researchers can effectively use E2-Tag in structural biology applications while minimizing potential artifacts or interference with native protein structure.

How can E2-Tag antibodies be utilized in the development of diagnostic assays?

E2-Tag antibodies offer powerful tools for developing sensitive and specific diagnostic assays with numerous methodological advantages:

Assay Design Principles:

• Blocking ELISA (bELISA) format:

  • Immobilize E2-tagged antigen on plate surface

  • Patient/sample antibodies compete with labeled anti-E2-Tag antibodies

  • Reduction in signal indicates presence of specific antibodies in sample

  • High specificity (>96%) and sensitivity (>97%) has been achieved with similar approaches

• Sandwich ELISA format:

  • Capture antibody binds target protein

  • E2-tagged detection antibody binds secondary epitope

  • Anti-E2-Tag antibody (enzyme-labeled) provides detection signal

  • Enhances specificity through dual-epitope recognition

Optimization Strategies:

• Antigen design:

  • Engineer recombinant antigens with strategically placed E2-Tags

  • Express multiple antigens with identical E2-Tags for multiplexed detection

  • Validate epitope accessibility in assay conditions

• Signal amplification:

  • Implement enzyme cascades for signal enhancement

  • Use nanoparticle-conjugated anti-E2-Tag antibodies

  • Explore electrochemical detection methods for quantitative results

• Validation protocols:

  • Test against panels of known positive and negative samples

  • Calculate sensitivity, specificity, and predictive values

  • Perform cross-reactivity studies with related pathogens

  • Establish reproducibility through inter-laboratory testing

Practical Applications:

• Infectious disease diagnostics:

  • Viral hepatitis serology

  • Arbovirus antibody detection

  • Parasitic disease diagnostics

• Veterinary diagnostics:

  • Similar to approaches used for CSFV E2 antibody detection

  • Herd immunity assessment

  • Surveillance programs

• Biomarker detection:

  • Cancer biomarkers

  • Autoimmune disease antibodies

  • Therapeutic drug monitoring

By standardizing the detection component of assays through E2-Tag technology, researchers can develop highly consistent diagnostic platforms with reduced development time and improved performance characteristics.

What are emerging applications of E2-Tag technology in advanced cellular imaging?

E2-Tag technology is expanding into sophisticated cellular imaging applications with several promising methodological developments:

Super-Resolution Microscopy:

• Site-specific labeling with small E2-Tags offers advantages over larger fluorescent protein fusions

• Combining E2-Tag with click chemistry approaches for orthogonal labeling strategies

• Development of high-affinity, photoswitchable fluorophore-conjugated anti-E2-Tag antibodies for STORM/PALM microscopy

• Implementation in multi-color super-resolution imaging for protein interaction studies

Live-Cell Imaging:

• Engineering of cell-permeable anti-E2-Tag antibody fragments (Fabs, nanobodies)

• Development of split-tag systems where E2-Tag complementation induces fluorescence

• Temporal control of E2-Tag expression for pulse-chase experiments

• Combination with optogenetic tools for spatiotemporal protein function analysis

Multiplexed Imaging:

• Cyclic immunofluorescence using E2-Tag as one of multiple epitope tags

• Mass cytometry (CyTOF) applications using metal-conjugated anti-E2-Tag antibodies

• Spatial transcriptomics integration with E2-Tag protein localization

• Correlated light and electron microscopy using E2-Tag for protein localization

Intrabody Applications:

• Engineering anti-E2-Tag antibody fragments for expression inside living cells

• Targeting specific subcellular compartments with E2-tagged proteins

• Modulating protein function through intrabody binding to E2-Tag

• Real-time monitoring of protein dynamics in living systems

These emerging applications represent the cutting edge of E2-Tag technology in cellular imaging, offering researchers powerful new tools for investigating protein function, localization, and dynamics at unprecedented resolution.

How might E2-Tag technology contribute to therapeutic protein development?

E2-Tag technology offers several methodological advantages in therapeutic protein development pipelines:

Manufacturing Process Development:

• Consistent purification protocols across diverse protein therapeutics

• High-affinity anti-E2-Tag antibody resins for efficient capture steps

• Site-specific tag placement to minimize impact on protein function

• Incorporation of protease cleavage sites for tag removal after purification

• Real-time monitoring of expression levels using anti-E2-Tag detection systems

Analytical Characterization:

• Standardized quantification assays across different therapeutic modalities

• Structural integrity assessment using conformation-sensitive anti-E2-Tag antibodies

• Batch-to-batch consistency evaluation with sensitive E2-Tag detection systems

• Development of reference standards with defined E2-Tag content

• Improved comparability studies between originator and biosimilar products

Functional Analysis:

• Receptor binding assays using E2-tagged ligands

• Cell-based potency assays with standardized detection systems

• Biodistribution studies using anti-E2-Tag antibodies for tissue localization

• Pharmacokinetic analysis with sensitive E2-Tag immunoassays

• Target engagement studies in preclinical models

Safety Assessment:

• Immunogenicity evaluation of E2-Tag in various contexts

• Development of anti-drug antibody assays based on E2-Tag detection

• Assessment of E2-Tag removal efficiency during manufacturing

• Cross-reactivity screening against human tissues

The strategic implementation of E2-Tag technology in therapeutic protein development can streamline manufacturing processes, enhance analytical capabilities, and provide consistent methodologies across diverse protein modalities, ultimately accelerating the path to clinical applications.