Glu-Glu Tag Monoclonal Antibody

What is the Glu-Glu epitope tag and what sequence does it recognize?

The Glu-Glu epitope tag represents a short peptide sequence commonly recognized as EYMPME (or alternatively EMYMPE). This tag derives from a portion of the polyomavirus middle T antigen with the original sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu, which plays a role in cellular transformation. Monoclonal antibodies targeting this epitope have been designed to specifically recognize this sequence when engineered into recombinant proteins . The tag's small size (six amino acids) makes it particularly valuable for minimizing interference with target protein function while enabling various immunological detection and purification approaches .

How does the Glu-Glu tag system compare to other epitope tagging systems?

The Glu-Glu tag offers several advantages compared to other tagging systems. Due to its small size (6 amino acids), it typically causes minimal interference with the structure and function of the tagged protein . Unlike some other systems such as the FLAG tag that has demonstrated limitations in nuclear protein detection (particularly in immunofluorescence applications), the Glu-Glu tag has shown reliable detection of nuclear proteins . This differential performance may be related to the abundance of arginine and lysine residues in nuclear proteins (>20% in histones), which potentially interfere with FLAG tag recognition . For researchers studying nuclear proteins, this makes the Glu-Glu tag system particularly valuable.

What are the optimal methods for introducing the Glu-Glu tag into a protein of interest?

Two standard approaches are commonly employed to introduce the Glu-Glu tag into proteins of interest:

• PCR-based oligonucleotide approach: This method utilizes oligonucleotides containing the Glu-Glu tag sequence (EYMPME) to amplify the gene of interest, ensuring in-frame expression. The tag sequence can be incorporated into either the forward or reverse primer, depending on whether N- or C-terminal tagging is desired .

• Expression vector system: Alternatively, researchers can clone their gene of interest into a vector already containing the Glu-Glu tag sequence. This approach simplifies the process and reduces the risk of PCR-introduced errors .

When designing your expression construct, critical factors include:

• Confirming in-frame fusion of the tag with the protein coding sequence

• Ensuring proper stop codon placement

• Strategic positioning of the tag (typically at N- or C-terminus) to minimize interference with protein function

What factors determine the optimal placement of the Glu-Glu tag in a protein construct?

The position of the Glu-Glu tag significantly impacts protein expression, function, and detection. Consider the following factors when determining tag placement:

• Functional domains: Avoid placing the tag near known functional domains, active sites, or binding interfaces that could interfere with protein activity.

• Protein termini accessibility: Choose between N- and C-terminal tagging based on the natural accessibility of these regions in your protein. For membrane proteins, consider which terminus faces the cytoplasm for improved detection.

• Protein localization signals: Be aware that tags may mask or interfere with endogenous localization signals (such as nuclear localization sequences or secretion signals) if placed at the terminus containing such signals .

• Folding considerations: Some proteins are sensitive to N-terminal modifications that can affect folding kinetics. In such cases, C-terminal tagging may be preferable .

How can detection sensitivity of Glu-Glu-tagged nuclear proteins be maximized in immunofluorescence applications?

Detecting nuclear proteins using epitope tags can be challenging due to the nuclear environment's properties. To maximize Glu-Glu-tagged nuclear protein detection:

• Fixation optimization: For nuclear proteins, a combination of paraformaldehyde fixation (2-4%) followed by permeabilization with 0.1-0.5% Triton X-100 often yields optimal results.

• Antibody selection: Choose high-affinity Glu-Glu monoclonal antibodies specifically validated for immunofluorescence applications. The search results indicate that G196 monoclonal antibody has demonstrated excellent nuclear protein detection in comparison studies .

• Blocking optimization: Use BSA or normal serum from the species of your secondary antibody to reduce background. For nuclear proteins, adding 5-10% normal serum can significantly improve signal-to-noise ratio.

• Signal amplification: For low-abundance nuclear proteins, consider tyramide signal amplification or use of highly sensitive fluorescent secondary antibodies.

The superior performance of the Glu-Glu tag system for nuclear protein detection is demonstrated in studies comparing it with the FLAG tag system. Researchers found that FLAG-tagged MRTF-B in cell nuclei stained more faintly compared to cytoplasmic protein, while the same protein with a Glu-Glu tag showed consistent detection in both compartments .

What protocols are recommended for purification of Glu-Glu-tagged proteins?

Purification of Glu-Glu-tagged proteins typically employs immunoaffinity chromatography with immobilized anti-Glu-Glu antibodies. The following protocol represents an optimized approach:

Materials:

• Anti-Glu-Glu monoclonal antibody coupled to agarose/sepharose beads

• Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitors

• Wash buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100

• Elution buffer options:
  a) Low pH: 0.1 M glycine, pH 2.5-3.0
  b) Peptide competition: 100-200 μg/ml synthetic Glu-Glu peptide
  c) Water-miscible organic solvents (less denaturing alternative)

Procedure:

• Prepare cell lysate in lysis buffer (1 hour at 4°C with gentle agitation)

• Clear lysate by centrifugation (15,000 × g, 15 min)

• Incubate cleared lysate with antibody-coupled beads (2-4 hours at 4°C with rotation)

• Wash beads 3-5 times with wash buffer

• Elute with chosen elution method:

  • For pH elution: apply glycine buffer, immediately neutralize with 1/10 volume of 1M Tris-HCl pH 8.0

  • For peptide elution: incubate with peptide solution for 1 hour at 4°C

  • For organic solvent elution: use water-miscible solvents as described in recent affinity tag systems

How can combinatorial tagging with Glu-Glu be implemented for multi-functional experimental approaches?

Combinatorial tagging—utilizing multiple tags on a single protein—offers powerful advantages for complex research applications. When implementing Glu-Glu tag in combinatorial approaches, consider these strategic options:

• Purification and visualization combinations: Pair Glu-Glu with a fluorescent protein tag (e.g., GFP) to enable both immunoaffinity purification and direct visualization.

• Sequential purification strategies: Combine Glu-Glu with another affinity tag (e.g., 6×His or MBP) positioned at the opposite terminus to enable tandem purification schemes, significantly enhancing purity for structural biology applications .

• Solubility enhancement: For proteins prone to aggregation or insolubility, combine Glu-Glu with solubility-enhancing tags such as MBP, SUMO, or Thioredoxin. This approach maintains the detection capabilities while improving protein expression and solubility .

• Cleavable tag designs: Incorporate a protease recognition sequence (e.g., TEV or PreScission) between the Glu-Glu tag and your protein of interest to enable tag removal after purification.

When designing combinatorial tag systems, careful consideration of tag positioning is critical. The following table outlines recommended configurations based on experimental goals:

This combinatorial approach enables researchers to capitalize on the unique advantages of multiple tagging systems while minimizing their individual limitations .

What are common cross-reactivity issues with Glu-Glu antibodies and how can they be addressed?

Cross-reactivity issues can complicate experiments using Glu-Glu tag antibodies. Analysis has shown that monoclonal antibodies against epitope tags can sometimes recognize endogenous proteins containing similar epitope sequences. For example, a search of the UniProtKB/Swiss-Prot database revealed potential cross-reactivity with 11 human proteins containing similar sequences to certain epitope tags .

To address potential cross-reactivity:

• Pre-adsorption testing: Before using Glu-Glu antibodies in a new cell line or tissue, conduct Western blot analysis of untransfected/wild-type samples to identify potential cross-reactive bands.

• Blocking with competing peptides: Include control samples where the antibody has been pre-incubated with excess synthetic Glu-Glu peptide to verify specificity of observed signals.

• Alternative detection methods: Complement antibody-based detection with other methods such as mass spectrometry to confirm protein identity.

• Multiple antibody validation: Use multiple anti-Glu-Glu antibodies from different sources or different clones to confirm specificity of detection.

• Negative controls: Always include untagged protein controls to establish baseline and differentiate specific from non-specific signals .

How can researchers troubleshoot low expression or detection of Glu-Glu-tagged proteins?

When facing challenges with low expression or poor detection of Glu-Glu-tagged proteins, consider the following methodical troubleshooting approach:

• Verify construct design:

  • Confirm the Glu-Glu tag is in-frame with your protein (sequence verification)

  • Check for proper stop codon placement

  • Ensure no mutations were introduced during cloning

• Expression optimization:

  • Test different expression conditions (temperature, induction time)

  • Consider codon optimization for the expression system

  • Evaluate alternative tag positions (N- vs C-terminal)

• Protein stability assessment:

  • Analyze protein half-life (cycloheximide chase)

  • Check for degradation products by Western blot

  • Add proteasome inhibitors to determine if low levels are due to degradation

• Detection enhancement:

  • Try different lysis buffers to improve protein extraction

  • Optimize antibody concentrations (typical working dilutions range from 1:300-5000 for Western blot)

  • Consider more sensitive detection methods (chemiluminescence vs. fluorescent)

• Subcellular localization considerations:

  • For nuclear proteins, use specialized nuclear extraction buffers

  • For membrane proteins, ensure proper solubilization with appropriate detergents

  • Consider if compartmentalization is reducing apparent expression levels

If expression issues persist despite these optimizations, consider potential fundamental issues such as protein toxicity, mRNA instability, or interference of the tag with protein folding .

What are the optimal storage and handling conditions for maintaining Glu-Glu tag antibody activity?

To maintain optimal Glu-Glu tag antibody performance over time:

• Storage temperature: Store antibodies at the recommended temperature, typically between 2-8°C for working solutions and -20°C for long-term storage of concentrated stocks .

• Buffer composition: Most commercial Glu-Glu antibodies are supplied in phosphate-buffered solution with preservatives such as 0.03% Thimerosal or 0.02% Proclin300, often with stabilizers like 50% Glycerol or 1% BSA .

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they can reduce antibody activity. Aliquot antibodies before freezing for single-use portions.

• Working dilution preparation: When preparing working dilutions, use fresh, cold buffer containing carrier protein (0.1-1% BSA) to prevent adsorption to tubes and maintain stability.

• Contamination prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination.

• Long-term storage considerations: For the Glu-Glu monoclonal antibody, storage at -20°C for up to one year is typically recommended, though specific products may have different optimal conditions .

Following manufacturer-specific storage recommendations is crucial, as formulations may vary between suppliers. Note that some manufacturers have updated storage recommendations from -20°C to 2-8°C for certain antibody formulations, highlighting the importance of checking product-specific guidelines .

How can Glu-Glu tag systems be optimized for studying protein-protein interactions?

For protein-protein interaction studies using Glu-Glu-tagged proteins:

• Co-immunoprecipitation optimization:

  • Use gentler lysis conditions to preserve protein-protein interactions (reduce detergent concentrations)

  • Consider crosslinking with membrane-permeable crosslinkers for transient interactions

  • Use appropriate controls to identify potential cross-reactive proteins in your system

• Bait protein considerations:

  • Position the Glu-Glu tag away from known interaction domains

  • Validate that the tagged protein maintains expected subcellular localization

  • Confirm biological activity of the tagged protein before interaction studies

• Proximity-based approaches:

  • Combine Glu-Glu tagging with proximity labeling methods (BioID, APEX) for identifying weak or transient interactors

  • Implement dual-tagging strategies where both potential interacting partners carry different tags for reciprocal validation

• Quantitative analysis:

  • Include appropriate negative controls (non-specific IgG, unrelated tagged protein)

  • Consider quantitative mass spectrometry approaches like SILAC or TMT labeling to distinguish specific from non-specific interactors

When employing Glu-Glu-tagged proteins for interaction studies in human cells, researchers should be aware of potential cross-reactions with endogenous proteins. The search results indicate that careful controls are necessary when using Glu-Glu-tagged proteins to search for binding partners, particularly in human cell systems .

How do the biochemical properties of the Glu-Glu tag influence experimental design for structural biology applications?

The biochemical properties of the Glu-Glu tag have significant implications for structural biology applications:

• Size and charge considerations:

  • The Glu-Glu tag contains multiple acidic residues, creating a negatively charged region that can affect protein behavior in certain buffer systems

  • At 6 amino acids (EYMPME), the tag is sufficiently small to minimize structural perturbations in most proteins

  • For crystallography studies, the small size reduces the likelihood of interfering with crystal packing

• Structural flexibility:

  • The tag likely adopts a flexible, unstructured conformation when attached to proteins

  • This flexibility can be advantageous for accessibility during antibody binding but may introduce disorder in X-ray crystallography

  • Consider incorporating a cleavable linker for removing the tag after purification but before crystallization attempts

• Buffer compatibility:

  • The acidic nature of the tag may affect protein behavior at pH values near the pKa of glutamic acid

  • For structural applications, test buffer systems with different pH values and ionic strengths to optimize protein stability

  • When using water-miscible organic solvents for elution, carefully evaluate the impact on protein structure

• NMR considerations:

  • For NMR studies, the additional residues from the tag will contribute signals that must be accounted for

  • The tag may introduce local chemical shift perturbations in nearby residues

  • Consider deuteration strategies if using the tag for proteins studied by NMR

Recent advancements in affinity tagging systems have demonstrated that structural knowledge of antibody-tag interactions can inform better tag design. For structural applications, researchers have successfully used concatenation of short recognition sequences to engineer tags with ideal solution binding kinetics, enabling efficient purification with non-denaturing elution conditions .

How does the Glu-Glu tag system compare with FLAG and other tag systems for nuclear protein detection?

Research has demonstrated significant differences in the performance of various epitope tag systems when detecting nuclear proteins. This comparative analysis highlights key findings:

• Nuclear protein detection efficiency:

  • Studies have shown that the FLAG tag system can be inadequate for immunofluorescence detection of nuclear proteins, with FLAG-tagged nuclear proteins staining notably more faintly than their cytoplasmic counterparts

  • In direct comparison studies, Glu-Glu-tagged nuclear proteins like NAC1 and ATF1 exhibited robust nuclear staining, demonstrating superior performance for nuclear localization studies

• Mechanistic basis for differential performance:

  • The high abundance of arginine and lysine in the nuclear environment (>20% of amino acids in histone proteins) may interfere with FLAG tag recognition

  • Arginine at mild pH can effectively dissociate FLAG-tagged proteins from anti-FLAG antibodies, potentially explaining the reduced nuclear detection efficiency

  • Nuclear proteins can constitute up to 55% of total nuclear content, creating a potentially interfering environment for certain tag systems

• Experimental validation:

  • When researchers changed from FLAG-tagged to Glu-Glu-tagged MRTF-B (a nuclear-cytoplasmic shuttle protein), they observed dramatically improved detection of the nuclear fraction

  • This allowed more accurate evaluation of relative subcellular distribution, critical for studies of protein trafficking and localization

These findings suggest that researchers studying nuclear proteins or proteins that shuttle between nucleus and cytoplasm should consider the Glu-Glu tag system as a preferred approach for immunofluorescence applications.

What advantages does the G196 epitope tag system offer compared to established tag systems?

While not a Glu-Glu tag system, the recently developed G196 epitope tag system offers instructive comparisons that inform tag selection decisions. The G196 system, based on a monoclonal antibody recognizing the five amino acid sequence Asp-Leu-Val-Pro-Arg, demonstrates several key advantages:

• High affinity and specificity:

  • The G196 mAb exhibits high binding affinity, as characterized by isothermal titration calorimetry

  • X-ray crystallography has been used to structurally analyze the variable regions, providing a molecular basis for its recognition properties

• Versatility across experimental systems:

  • The G196 system has been successfully applied to both human cells and yeast systems

  • It effectively detected nuclear reporter proteins in both contexts, including the transcription regulator NAC1 in human cells and the Atf1/Pcr1 heterodimeric transcription factor in yeast

• Nuclear protein detection capabilities:

  • Similar to the Glu-Glu tag, the G196 system shows superior performance in detecting nuclear proteins compared to the FLAG tag system

  • This positions both G196 and Glu-Glu tag systems as valuable alternatives when studying nuclear or nucleocytoplasmic shuttling proteins

Understanding the comparative advantages of different tagging systems allows researchers to make informed choices based on their specific experimental requirements, target protein properties, and cellular localization patterns.