Myc-Tag Monoclonal Antibody

What is a Myc-tag and what is its amino acid sequence?

The Myc-tag is a short peptide sequence derived from the human c-Myc protein, with the amino acid sequence EQKLISEEDL (10 amino acids). This peptide was originally identified as the epitope recognized by the monoclonal antibody 9E10 developed against human c-Myc protein. The tag can be genetically fused to proteins of interest, allowing for detection and purification of recombinant proteins without the need for protein-specific antibodies. The Myc-tag is widely used in molecular biology as it is relatively small and typically does not interfere with protein function when strategically placed .

How do Myc-tag monoclonal antibodies work in protein detection systems?

Myc-tag monoclonal antibodies function through specific recognition of the Myc peptide sequence that has been genetically fused to a protein of interest. When a Myc-tag is incorporated into a recombinant protein (either at the N-terminus, C-terminus, or internally), the antibody binds to this epitope with high specificity. The antibody-epitope interaction forms the basis for various detection methods including Western blotting, immunoprecipitation, immunofluorescence, and flow cytometry. The specificity of this interaction allows researchers to isolate, visualize, and quantify the tagged protein from complex biological samples without requiring protein-specific antibodies .

How does epitope positioning affect Myc-tag antibody recognition?

The position of the Myc-tag within a fusion protein significantly impacts antibody recognition in experimental applications. Research has demonstrated that all commonly used Myc-tag antibodies display context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. This variability is most pronounced with the legacy antibody 9E10, which exhibits high context-dependent detection variability across different experimental conditions. The recognition efficiency is influenced by both the terminal placement of the tag (N- versus C-terminal) and the neighboring amino acid sequences that can affect epitope accessibility and conformation. Modern purpose-made monoclonal antibodies such as 4A6 and 9B11 demonstrate much less context sensitivity, providing more consistent detection across different tagging contexts . Researchers should consider carefully which antibody clone to use based on their specific tagging strategy and validation experiments with their particular fusion protein.

What controls should be included when using Myc-tag antibodies in Western blotting?

When conducting Western blotting with Myc-tag antibodies, several critical controls should be incorporated to ensure experimental validity and accurate interpretation of results:

• Positive control: Include a well-characterized Myc-tagged protein with known molecular weight and expression level. This confirms antibody functionality and provides a reference signal intensity.

• Negative control: Use untransfected cells or cells expressing the untagged protein to identify any non-specific binding.

• Knockout/knockdown control: Where available, include samples from cell lines where the endogenous c-Myc has been knocked out to identify potential cross-reactivity, as demonstrated in validation studies with HEK-293T MYC CRISPR-Cas9 edited cells .

• Loading control: Include antibodies against housekeeping proteins (such as GAPDH or β-actin) to normalize protein loading across lanes.

• Concentration gradient: Testing a dilution series of both antibody concentration and protein sample can help determine optimal detection conditions and assess sensitivity limits, as demonstrated in analytical studies with THE™ c-Myc Tag Antibody .

These controls collectively help distinguish true signals from artifacts and provide benchmarks for quantitative comparisons across experiments.

What is the optimal working concentration for Myc-tag antibodies in various applications?

The optimal working concentration for Myc-tag antibodies varies significantly depending on the specific application, antibody clone, and experimental context. Based on empirical data from sensitivity analyses:

For optimal results, researchers should perform a titration experiment with their specific protein and antibody combination. Signal development conditions (e.g., IRDye™ 800 Conjugated secondary antibodies versus chemiluminescence) will also affect the optimal primary antibody concentration .

How do neighboring sequences affect the recognition of Myc-tag by different monoclonal antibodies?

The influence of neighboring sequences on Myc-tag recognition represents a significant technical challenge in epitope tagging systems. Comprehensive studies comparing multiple Myc-tag antibodies have revealed that sequence context critically impacts epitope accessibility and recognition. The 9E10 clone, despite being the most cited Myc-tag antibody, exhibits remarkably high context-dependent detection variability, as confirmed by both Western blotting experiments and peptide microarray analyses. This variability occurs because amino acid sequences flanking the Myc epitope can alter epitope conformation, create steric hindrance, or modify charge distribution at the binding interface .

In contrast, newer purpose-developed antibodies such as 4A6 and 9B11 demonstrate substantially reduced context sensitivity, providing more uniform reactivity across diverse fusion protein constructs. These next-generation antibodies were specifically engineered to minimize the impact of neighboring sequences. For researchers studying novel proteins or working with challenging constructs, selecting less context-sensitive antibodies can dramatically improve detection consistency and experimental reproducibility . When designing new Myc-tagged constructs, including flexible linker sequences (such as Gly-Ser repeats) between the tag and the protein of interest can reduce context effects by isolating the epitope from potentially interfering protein structures.

What strategies can minimize context-dependent variability when using Myc-tag antibodies?

To overcome context-dependent variability when using Myc-tag antibodies, researchers can implement several strategic approaches:

• Selection of optimal antibody clone: Choose newer purpose-made monoclonal antibodies like 4A6 or 9B11 that have been specifically developed to exhibit reduced context sensitivity compared to the legacy 9E10 clone .

• Linker sequence incorporation: Include flexible glycine-serine linkers (e.g., GGGGS) between the Myc-tag and the protein of interest to reduce steric hindrance and conformational constraints.

• Multi-epitope tagging: Implement tandem Myc-tags (2-3 repeats) to increase avidity and detection sensitivity, which can overcome partial epitope masking effects.

• Position optimization: Test both N-terminal and C-terminal tagging configurations to identify the optimal position for efficient antibody recognition of your specific protein.

• Validation across multiple detection methods: Confirm protein expression using complementary techniques (e.g., Western blot and immunofluorescence) as context sensitivity can vary between applications.

• Alternative epitope systems: For proteins where Myc-tag detection remains problematic despite optimization, consider alternative tagging systems such as FLAG, HA, or G196 (DLVPR) .

These approaches, especially when used in combination, can significantly reduce variability and improve detection consistency across different experimental contexts.

How do different Myc-tag monoclonal antibodies compare in terms of sensitivity and specificity?

Comparative analyses of various Myc-tag monoclonal antibodies reveal significant differences in sensitivity and specificity profiles that can dramatically impact experimental outcomes:

Sensitivity testing with THE™ c-Myc Tag Antibody has demonstrated detection capabilities at dilutions as extreme as 1:20,000 (equivalent to 0.05 μg/ml), significantly outperforming the traditional 9E10 clone in Western blotting applications . Meanwhile, context sensitivity testing has established that 4A6 and 9B11 provide more uniform detection of Myc-tagged proteins regardless of tag position or neighboring sequences .

When selecting an antibody for specific applications, researchers should consider these performance characteristics alongside the particular requirements of their experimental system.

What are common causes of weak or absent signals when using Myc-tag antibodies?

When researchers encounter weak or absent signals with Myc-tag antibodies, several factors may be responsible:

• Context-dependent epitope masking: The surrounding protein structure may obscure the Myc epitope, particularly with the 9E10 clone which shows high context sensitivity. This is especially problematic for proteins with complex tertiary structures or when the tag is positioned near structured domains .

• Insufficient expression levels: The tagged protein may be expressed at levels below the detection threshold. This is particularly common with unstable proteins or inefficient expression systems.

• Protein degradation: Rapid turnover of the fusion protein can result in low steady-state levels. Proteasome inhibitors (e.g., MG132) can help determine if degradation is occurring.

• Tag cleavage: Proteolytic removal of the tag during expression or sample preparation will eliminate the epitope. Adding protease inhibitors to lysis buffers is essential.

• Incompatible fixation methods: For immunofluorescence applications, certain fixation protocols may alter epitope structure. Different fixatives (PFA versus methanol) should be tested.

• Antibody batch variability: Quality inconsistencies between antibody lots can affect performance. Including positive controls helps identify antibody-related issues.

• Cross-reactivity with endogenous proteins: Some cell lines express proteins that may cross-react with Myc-tag antibodies, potentially leading to confounding results .

Systematic troubleshooting of these factors through appropriate controls and optimization steps can resolve most detection problems.

How can immunofluorescence protocols using Myc-tag antibodies be optimized?

Optimizing immunofluorescence protocols for Myc-tag antibodies requires attention to several critical parameters:

• Fixation method selection: Compare paraformaldehyde (most common, 4% for 15-20 minutes) versus methanol fixation (100%, -20°C for 10 minutes) to determine which best preserves the Myc epitope in your specific construct. Some Myc-tagged proteins show dramatically different detection efficiency depending on fixation method.

• Permeabilization optimization: Test mild (0.1% Triton X-100) versus stronger (0.5% Triton X-100 or 0.1% SDS) permeabilization reagents to ensure antibody access to the epitope while preserving cellular structures.

• Blocking buffer composition: Incorporate both serum (5-10%) and BSA (1-3%) in blocking solutions to reduce both specific and non-specific background binding.

• Antibody selection: For nuclear proteins, certain anti-FLAG antibodies perform poorly for immunofluorescence of nuclear targets, suggesting that antibody selection is particularly important for nuclear Myc-tagged proteins .

• Signal amplification: For low-abundance proteins, consider using biotinylated secondary antibodies with fluorescent streptavidin for signal amplification, or tyramide signal amplification systems.

• Mounting media selection: Use anti-fade mounting media containing DAPI for nuclear counterstaining and prolonged fluorescence stability during imaging.

• Positive controls: Include cells expressing well-characterized Myc-tagged proteins known to work in immunofluorescence to validate protocol conditions.

Following these optimization strategies can significantly improve detection sensitivity and specificity in immunofluorescence applications with Myc-tagged proteins.

What considerations should be made when using Myc-tag in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) experiments using Myc-tag antibodies require careful consideration of several factors to ensure reliable results:

• Potential cross-reactivity: The Myc-tag sequence (DLVPR) appears in 11 human proteins according to UniProtKB/Swiss-Prot database searches. When conducting co-IP experiments to identify binding partners, researchers must exercise caution as these endogenous proteins could be inadvertently precipitated and misidentified as interaction partners .

• Binding buffer composition: The choice of detergents (NP-40, Triton X-100, or CHAPS) and salt concentration critically affects the preservation of protein-protein interactions. Mild conditions (0.1% NP-40, 150mM NaCl) are recommended for initial experiments, with optimization for specific protein complexes.

• Antibody immobilization strategy: Compare different immobilization approaches (pre-binding to Protein G beads versus direct immunoprecipitation) to determine which method provides optimal pulldown efficiency while minimizing background.

• Pre-clearing lysates: Implementing a pre-clearing step with isotype control antibodies can significantly reduce non-specific protein binding to beads or antibodies.

• Negative controls: Include IPs with non-expressing cells, isotype control antibodies, and untagged protein controls to identify non-specific interactions.

• Elution conditions: Optimize elution conditions (competitive elution with Myc peptide versus SDS elution) based on downstream applications and the nature of the protein interactions being studied.

• Validation with reciprocal co-IP: Confirm interactions by performing reverse co-IP experiments using antibodies against the putative interacting partner to precipitate the Myc-tagged protein.

These considerations help ensure that co-IP results represent genuine protein-protein interactions rather than experimental artifacts.

What are the advantages and limitations of using Myc-tag compared to other epitope tag systems?

Myc-tag offers distinct advantages and limitations compared to other popular epitope tagging systems:

The choice between these systems depends on the specific experimental requirements. For proteins where structure and function are particularly sensitive to modifications, the smaller G196-tag may be preferable. For applications requiring highly efficient purification, FLAG-tag often performs better. When working with nuclear proteins, researchers should be aware that anti-FLAG antibody M2 has been reported to perform poorly in immunofluorescence detection of nuclear proteins .

The Myc-tag system, particularly when used with newer antibodies like 4A6 and 9B11 that show reduced context sensitivity, offers a good balance of small size and reliable detection across multiple applications . This versatility has contributed to its widespread adoption in molecular biology research.

How are recent innovations improving the utility of Myc-tag antibodies?

Recent innovations in Myc-tag antibody technology are substantially enhancing their research utility:

• Development of context-independent antibodies: Newer monoclonal antibodies such as 4A6 and 9B11 have been specifically engineered to exhibit much more uniform reactivity across diverse assays and reduced context sensitivity compared to the legacy 9E10 antibody. These purpose-made antibodies provide more consistent detection regardless of tag position or neighboring sequences .

• Recombinant antibody technologies: The introduction of recombinant Myc-tag binding proteins and nanobodies (such as Myc-Trap® or Myc VHH) offers advantages in terms of reproducibility, defined composition, and reduced batch-to-batch variation compared to traditional hybridoma-produced antibodies .

• Combination with emerging techniques: Integration of Myc-tag systems with proximity labeling methods (BioID, APEX) is expanding their utility for identifying transient or weak protein interactions in living cells.

• High-throughput screening compatibility: Optimization of Myc-tag antibodies for automated imaging platforms and high-content screening systems is facilitating large-scale protein localization and interaction studies.

• Dual-tag strategies: Development of optimized protocols for sequential purification using Myc-tag in combination with other epitope tags (such as FLAG or HA) is enabling isolation of highly pure protein complexes while maintaining native conditions.

These innovations are collectively extending the applications of Myc-tag antibodies beyond traditional uses, making them increasingly valuable tools for cutting-edge research in proteomics, interactomics, and structural biology.