Avi-Tag Monoclonal Antibody

What is an Avi-Tag and how do monoclonal antibodies recognize it?

The Avi-Tag is a 15-amino acid peptide sequence (GLNDIFEAQKIEWHE) that can be genetically encoded onto a protein of interest. This sequence serves as a substrate for the biotin ligase enzyme BirA from E. coli, which covalently attaches biotin to the specific lysine residue within the tag . Monoclonal antibodies against Avi-Tag have been developed with varying specificities - some recognize only the non-biotinylated form, some only the biotinylated form, and others recognize the tag regardless of biotinylation status. This diversity allows researchers to select antibodies appropriate for their specific experimental needs .

How does the AviTrap system differ from standard Avi-Tag antibody applications?

The AviTrap system utilizes a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. This specificity allows for efficient separation of biotinylated from non-biotinylated proteins. After a brief 10-minute incubation with antibody-conjugated resin, non-biotinylated AviTagged proteins are trapped while fully biotinylated material freely passes through. This provides a simple one-step purification solution for enriching biotinylated AviTagged proteins, addressing the common challenge of incomplete biotinylation reactions . This approach is particularly valuable when working with proteins that require complete biotinylation for downstream applications.

What are the typical applications for Avi-Tag monoclonal antibodies in protein research?

Avi-Tag monoclonal antibodies serve multiple functions in protein research:

These antibodies are particularly valuable for detecting recombinant proteins with Avi-Tags across diverse experimental platforms, allowing for consistent tracking throughout multi-step experimental workflows .

How can I optimize Avi-Tag monoclonal antibody detection specificity when working with complex protein mixtures?

Optimizing specificity requires careful consideration of several factors:

• Antibody selection: Choose clones with demonstrated specificity for your application. For example, clone W19258A recognizes Avi-tagged proteins regardless of biotinylation status, whereas other antibodies like those used in AviTrap specifically recognize only non-biotinylated tags .

• Blocking optimization: Use 3-5% BSA in TBS-T for Western blots to minimize non-specific binding, particularly when working with complex protein mixtures.

• Cross-reactivity testing: Include appropriate controls in your experimental design, such as proteins with other common tags (e.g., His-Tag). Research shows that high-quality Avi-Tag antibodies like clone 2623F demonstrate no cross-reactivity with His-tagged proteins in Western blots .

• Titration experiments: Antibody concentration should be titrated for each specific Avi-tagged protein of interest. As noted in application guidelines: "Antibody concentration per application is subject to the Avi-tag-recombinant protein. Titration is required for every specific protein" .

• Secondary antibody selection: Match the isotype of your primary antibody (e.g., IgG2a for many Avi-Tag antibodies) with an appropriate secondary antibody to maximize signal-to-noise ratio.

What strategies can improve the in vivo biotinylation efficiency of Avi-Tagged proteins for subsequent antibody-based applications?

Efficient biotinylation is critical for many downstream applications. Research indicates several optimization strategies:

• Expression system selection: The AVB101 cell line contains a chromosomally integrated copy of the birA gene, providing consistent in vivo biotinylation . This system has demonstrated robust performance in nanobody-AviTag fusion protein production.

• Biotin concentration optimization: Supplementing culture media with 50 μM D-biotin during protein expression enhances biotinylation efficiency.

• Co-expression optimization: When using systems that require co-transfection with BirA, maintain a 1:3 ratio of target protein to BirA plasmids to ensure sufficient biotin ligase activity.

• Temperature adjustment: Reducing induction temperature to 25°C can improve folding and biotinylation of complex proteins.

• Post-purification assessment: Use dot immunoassay with SA-HRP (streptavidin-horseradish peroxidase) at 0.3 μg/mL to verify biotinylation efficiency before proceeding to antibody-based applications .

For proteins that remain incompletely biotinylated, implementing the AviTrap system can effectively separate the biotinylated fraction for downstream applications .

How do organic solvents affect Avi-Tag antibody binding kinetics and what implications does this have for experimental design?

Research on nanobody-AviTag fusion proteins has revealed significant solvent-dependent effects that should inform experimental design:

• Methanol effects: Binding activity decreases to approximately 63% at 2.5% methanol concentration but paradoxically increases to 83% at 10% methanol before declining significantly (22% activity) at 60% concentration .

• Acetonitrile tolerance: Notably, acetonitrile shows enhanced compatibility, with binding activity maintained above 115% at concentrations between 2.5-20% .

• Mechanism consideration: These solvent effects likely influence the three-dimensional conformation of the antibody-antigen binding interface rather than directly disrupting primary binding interactions.

• Experimental implications: When designing extraction protocols for Avi-Tagged proteins from complex matrices (particularly for small molecule detection applications like ochratoxin A analysis), acetonitrile may be preferred over methanol as an extraction solvent .

• Application-specific optimization: For each application, especially those involving sample preparation with organic solvents, binding activity should be empirically determined across a range of solvent concentrations.

What are the optimal buffer conditions for using Avi-Tag monoclonal antibodies in Western blot applications?

Based on validated protocols from multiple sources, these buffer conditions maximize sensitivity and specificity:

• Transfer buffer: 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3 for standard proteins. For high molecular weight proteins, reducing SDS to 0.05% can improve transfer efficiency.

• Blocking solution: 5% non-fat dry milk or 3% BSA in TBS-T (TBS + 0.05% Tween-20) for 1 hour at room temperature .

• Primary antibody dilution: Most Avi-Tag antibodies perform optimally at 0.1-1 μg/mL in blocking buffer. Clone-specific recommendations range from 1:1000 to 1:640000 dilution depending on the antibody .

• Washing: Five washes with TBS-T for 5 minutes each following both primary and secondary antibody incubations.

• Secondary antibody: HRP-conjugated anti-mouse IgG (for mouse monoclonal antibodies) or anti-rabbit IgG (for rabbit monoclonal antibodies) at 1:5000-1:10000 dilution .

• Detection system: Enhanced chemiluminescence substrates are typically recommended, with exposure times ranging from 30 seconds to 5 minutes depending on protein abundance.

How should I design control experiments when using Avi-Tag monoclonal antibodies for novel recombinant protein characterization?

Robust experimental design requires comprehensive controls:

• Positive controls: Include a well-characterized Avi-tagged protein such as Maltose Binding Protein-AviTag (MBP-AviTag) or VEGF-165-AviTag, which have been validated in multiple antibody characterization studies .

• Negative controls:

  • Non-tagged version of your protein of interest

  • Protein with alternative tag (e.g., His-tag) to confirm specificity

  • For cell-based assays, include untransfected cells as demonstrated in flow cytometry and immunocytochemistry applications

• Biotinylation controls: When assessing biotinylation, include both biotinylated and non-biotinylated versions of the same Avi-tagged protein. Commercial kits like BIS-300 contain fully biotinylated and unbiotinylated MBP-AviTag as standards .

• Cross-reactivity assessment: Test antibody against proteins with similar tag sequences or endogenous proteins with potential epitope mimicry.

• Method validation: When implementing a new detection method, validate results using orthogonal approaches (e.g., mass spectrometry) as demonstrated in AviTrap development studies .

What are the critical parameters for successful application of Avi-Tag monoclonal antibodies in structural biology research?

Structural biology applications require particular attention to:

• Antibody selection: For structural studies, use antibodies validated in cryo-EM applications. Research has demonstrated successful incorporation of Avi-Tags in SARS-CoV-2 spike protein structural studies .

• Construct design considerations:

  • N-terminal vs. C-terminal tagging: Some antibodies specifically recognize C-terminal AviTags , while others work with both orientations. Placement can affect protein folding and function.

  • Linker optimization: Incorporate flexible linkers (e.g., 10-residue linkers) between the protein of interest and the AviTag to minimize steric hindrance .

  • Protease cleavage sites: Include HRV3C protease recognition sites to enable tag removal for crystallization studies while maintaining biotin-mediated purification capability .

• Expression system: Transient transfection yields vary significantly by protein size - from ~0.5 mg/L for large proteins like spike ectodomain to >5 mg/L for smaller domains . Scale expression systems accordingly.

• Purification strategy: Consider tag-based purification with on-column biotinylation using the following workflow:

  • Capture via N-terminal purification tag (e.g., single-chain Fc)

  • On-column biotinylation

  • Elution via specific protease treatment rather than low pH to preserve native conformation

• Structural verification: Following purification, verify structural integrity using techniques like size-exclusion chromatography and negative-stain electron microscopy before proceeding to high-resolution structural determination.

How can I troubleshoot inconsistent signal intensity when using Avi-Tag monoclonal antibodies in immunoassays?

Inconsistent signal intensity often stems from several key factors:

• Epitope accessibility: The Avi-Tag sequence (GLNDIFEAQKIEWHE) may be partially obscured depending on protein conformation. Research indicates that:

  • C-terminal tags are often more accessible than internal tags

  • Incorporation of flexible linkers (≥10 amino acids) improves antibody recognition

  • Denaturation conditions in Western blots typically provide better epitope exposure than native conditions in immunoprecipitation

• Biotinylation heterogeneity: Incomplete biotinylation creates mixed populations that affect signal consistency. Solutions include:

  • Implementing the BRTA assay to determine biotinylation extent

  • Using AviTrap to purify the fully biotinylated fraction

  • Optimizing in vivo biotinylation conditions as detailed previously

• Antibody-specific variables:

  • Storage conditions: Most Avi-Tag antibodies should be stored at 4°C and not subjected to freeze-thaw cycles

  • Dilution buffer composition: Adding 0.2% BSA and 0.05% sodium azide to PBS improves stability

  • Lot-to-lot variation: Validate each new lot against a reference standard

• Technical considerations:

  • Incubation temperature: Room temperature typically provides optimal binding kinetics

  • Incubation time: Extending from standard 1-hour to overnight at 4°C can improve signal for low-abundance proteins

  • Washing stringency: Excessive washing can reduce signal; optimize wash buffer composition based on signal-to-noise ratio

What considerations are important when using Avi-Tag monoclonal antibodies for multi-color flow cytometry experiments?

Multi-color flow cytometry with Avi-Tag antibodies requires careful experimental design:

• Fluorophore selection: When using directly conjugated antibodies, consider:

  • Alexa Fluor 488 anti-Avi-tag typically shows best performance for FITC channel applications

  • Alexa Fluor 647 anti-Avi-tag provides optimal separation in the APC channel

  • Avoid PE conjugates if biotinylation status authentication is required, as they may interfere with biotin detection

• Compensation considerations:

  • Avi-Tag antibodies may demonstrate broader emission spectra than typical immunophenotyping antibodies

  • Include single-color controls for each Avi-Tag antibody conjugate

  • Use antibody capture beads rather than cells for creating compensation matrices

• Protocol optimization:

  • Cell fixation significantly impacts epitope accessibility; 2% paraformaldehyde provides optimal preservation

  • Permeabilization (if needed) should use 0.1% saponin rather than harsher detergents

  • Antibody concentration requires titration for each application; ≤0.125 μg per million cells in 100 μL volume is recommended as a starting point

• Controls for Avi-Tagged proteins:

  • Include untransfected cells as negative controls

  • For dual-color experiments, include FMO (fluorescence minus one) controls

  • When studying membrane proteins, compare surface versus intracellular staining to assess trafficking efficiency

How are Avi-Tag antibodies being integrated into advanced biotechnology platforms?

Recent research demonstrates increasing integration of Avi-Tag antibodies into cutting-edge platforms:

• Single-cell protein analysis: Avi-Tag antibodies are being incorporated into CyTOF (mass cytometry) panels for high-dimensional single-cell protein profiling, enabling simultaneous detection of Avi-tagged proteins alongside cellular markers.

• Spatial proteomics: Integration with multiplexed ion beam imaging (MIBI) allows for spatial localization of Avi-tagged proteins within tissue architecture at subcellular resolution.

• Proximity labeling applications: Avi-Tag antibodies can be used to validate BioID or APEX2 proximity labeling experiments by confirming the presence of biotinylated proteins using orthogonal detection methods.

• Vaccine development: The SARS-CoV-2 research demonstrates the utility of Avi-Tag systems in rapidly developing biotinylated molecular probes for vaccine research, with yields ranging from ~0.5 mg/L for complete spike ectodomain to >5 mg/L for subdomains .

• Microfluidic systems: Integration with droplet-based single-cell analysis platforms facilitates high-throughput screening of Avi-tagged protein variants.

What advances in protein engineering are enhancing the utility of Avi-Tag antibody detection systems?

Protein engineering is expanding Avi-Tag system capabilities:

• Enhanced tag variants: Engineering of the AviTag sequence itself has produced variants with:

  • Improved biotinylation kinetics

  • Reduced immunogenicity for in vivo applications

  • Site-specific incorporation via non-canonical amino acid technology

• Antibody engineering advances:

  • THIOMAB format with S400C mutated cysteine enables site-specific conjugation to resins for enhanced purification applications

  • Single-domain antibody (nanobody) fusions with AviTag demonstrate superior performance in certain applications, particularly for small molecule detection

  • COSMO (Comprehensive Substitution for Multidimensional Optimization) approaches have generated antibody variants with dramatically improved binding properties

• Multifunctional fusion proteins:

  • Single chain-Fc (scFc) with "knob-in-hole" features prevent dimer formation

  • Integration of AviTag with HRV3C protease recognition sites enables tag removal while maintaining biotinylation capability

  • Combination of AviTag with split protein complementation systems for detecting protein-protein interactions in living cells