Myc tag Monoclonal Antibody

What is the Myc tag and why is it used in research?

The Myc tag is a short synthetic polypeptide sequence derived from the human c-myc protein, specifically corresponding to amino acids 410-419 with the sequence EQKLISEEDL. This tag is widely used in molecular biology research when an antibody against the protein of interest is not available. By fusing the Myc tag to a protein of interest through recombinant DNA technology, researchers can track, purify, and study the tagged protein using commercially available anti-Myc tag antibodies . This approach is particularly valuable for newly discovered proteins or when generating specific antibodies is challenging or time-consuming.

What are the primary applications of Myc tag monoclonal antibodies?

Myc tag monoclonal antibodies have diverse applications in molecular and cellular biology research:

These applications enable researchers to study protein expression, localization, interaction, and function in various experimental systems .

What is the molecular weight of the Myc tag and how does it affect tagged proteins?

The Myc tag has a molecular weight of approximately 1203 Da, comprising the ten amino acid sequence EQKLISEEDL . When designing experiments, researchers should account for this additional mass when analyzing tagged proteins. While the small size of the Myc tag generally minimizes interference with protein structure and function, it's advisable to validate that the tag does not affect the biological activity of your protein of interest through appropriate controls. The tag can be fused to either the N-terminus or C-terminus of the target protein, depending on which arrangement is less likely to interfere with protein function .

How do different Myc tag monoclonal antibody clones compare in performance?

A comprehensive analysis of six antibodies recognizing the c-Myc epitope tag revealed significant differences in context-dependent detection capabilities:

Research has demonstrated that recently developed, purpose-made monoclonal antibodies (particularly 4A6 and 9B11) have much more uniform reactivity in diverse assays and are much less context-sensitive than the legacy antibody 9E10 . This finding is important when selecting antibodies for experiments where the Myc tag may be positioned in different contexts.

How does the position of the Myc tag affect antibody recognition?

Epitope tag position and neighboring sequences can significantly affect recognition by antibodies. Context-dependent differences in tag binding may have wide-ranging effects on data interpretation. Western blotting tests have shown that all Myc tag antibodies display some level of context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins, though to varying degrees .

For optimal detection:

• Consider testing multiple anti-Myc antibody clones if you observe weak or inconsistent signals

• Be aware that clone 9E10, despite being widely cited, shows high context-dependent variability

• For applications requiring consistent detection regardless of tag position, consider clones 4A6 or 9B11

• When possible, standardize the position of the tag (N- or C-terminal) across all constructs in a study

This context sensitivity was confirmed through peptide microarray analyses, demonstrating that epitope accessibility plays a crucial role in antibody binding efficiency .

What are the optimal storage conditions for Myc tag monoclonal antibodies?

To maintain antibody activity and stability:

• Store unopened antibodies at -20°C for up to one year

• Make small aliquots to avoid repeated freeze-thaw cycles

• Prior to opening, briefly spin tubes to ensure liquid collection at the bottom

• For working solutions, store at 4°C for short-term use (2-4 weeks)

• Protect conjugated antibodies (HRP, FITC) from light exposure

• Follow manufacturer's recommendations for storage buffer composition (typically PBS, pH 7.4, with 0.05% sodium azide and sometimes glycerol or BSA)

Proper storage significantly impacts experimental reproducibility and antibody shelf-life. Degradation can lead to reduced sensitivity, increased background, and experiment failure.

How should dilution factors be optimized for different experimental applications?

Optimal dilution factors vary by application and specific antibody clone. While manufacturers provide recommended ranges, optimization for your specific experimental system is crucial:

• Western Blot optimization:

  • Start with the recommended dilution (typically 1:1000)

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Evaluate signal-to-noise ratio at each dilution

  • Select the dilution that provides clear specific bands with minimal background

• Immunofluorescence optimization:

  • Begin with manufacturer's recommendation (typically 1:200-1:800)

  • Test on cells with known expression levels of Myc-tagged proteins

  • Assess specificity by comparing transfected vs. non-transfected cells

  • Minimize autofluorescence with appropriate controls and blocking reagents

• Immunoprecipitation optimization:

  • Antibody amount should be proportional to protein concentration

  • Typically use 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  • Optimize binding conditions (time, temperature, buffer composition)

  • Include appropriate negative controls (non-specific IgG, non-transfected cells)

Remember that exact working dilution needs to be determined by the end user for each specific experimental setup .

How can potential artifacts from Myc tagging be identified and mitigated?

Despite its utility, Myc tagging can potentially introduce artifacts that require careful consideration:

• Protein mislocalization:

  • Compare localization with alternative tags (FLAG, HA)

  • Use both N- and C-terminal tagged versions

  • Validate with endogenous protein localization when possible

  • Consider disruption of localization signals (NLS, NES, transmembrane domains)

• Altered protein function:

  • Perform functional assays comparing tagged and untagged versions

  • If available, compare with endogenous protein activity

  • Consider using a cleavable linker between tag and protein of interest

  • Test both N- and C-terminal tags to identify optimal configuration

• Expression level artifacts:

  • Use inducible expression systems to control expression levels

  • Compare multiple clones with varying expression levels

  • Include dose-response experiments in functional assays

  • Consider single-cell analyses to account for expression heterogeneity

• Aggregation or stability issues:

  • Assess protein solubility and stability through biochemical fractionation

  • Examine protein half-life with cyclohexamide chase experiments

  • Consider native PAGE alongside denaturing conditions

  • Evaluate oligomerization state with size exclusion chromatography

The small size of the Myc tag (10 amino acids) generally minimizes interference with protein function, but validation is essential for each new protein under investigation.

What strategies can resolve contradictory results from different Myc tag antibody clones?

When different antibody clones yield conflicting results, systematic troubleshooting is necessary:

• Context-dependent epitope accessibility:

  • The high context sensitivity of some clones (particularly 9E10) may explain detection discrepancies

  • Try multiple antibody clones with different epitope recognition characteristics

  • Clone 4A6 or 9B11 may provide more consistent detection across contexts

  • Consider using a dual-tagging approach (e.g., Myc+FLAG) for validation

• Methodological approach:

  • Compare native versus denaturing conditions

  • Assess fixation effects in microscopy applications

  • Optimize blocking conditions to reduce non-specific binding

  • Consider epitope retrieval methods for fixed samples

• Systematic comparison:

  • Create a standardized experimental system with known positive controls

  • Test all antibody clones side-by-side under identical conditions

  • Quantify detection efficiency using recombinant standards

  • Document batch-to-batch variation from the same manufacturer

• Independent validation:

  • Verify results with orthogonal methods (mass spectrometry, alternate tags)

  • Use CRISPR/Cas9 knock-in to tag endogenous proteins

  • Consider proximity labeling approaches (BioID, APEX) as complementary methods

  • Validate with functional assays that don't rely on antibody detection

Understanding the specific binding characteristics of each antibody clone can help reconcile apparently contradictory results and guide experimental design.

How can multiplexed detection of Myc-tagged proteins be optimized alongside other epitope tags?

Simultaneous detection of multiple tagged proteins requires careful experimental design:

• Compatible tag selection:

  • Choose tags with minimal cross-reactivity (Myc, FLAG, HA, V5)

  • Ensure antibodies are raised in different host species to enable secondary antibody discrimination

  • Consider size differences between tags for distinguishing proteins of similar molecular weight

  • Test for potential tag-tag interactions in your experimental system

• Multi-color immunofluorescence optimization:

  • Select fluorophores with minimal spectral overlap

  • Perform single-color controls to establish detection parameters

  • Use sequential staining protocols for antibodies from the same species

  • Apply appropriate controls for background autofluorescence and bleed-through

• Sequential immunoprecipitation strategies:

  • Perform tandem IP using different tags to verify protein-protein interactions

  • Elute specifically using competitor peptides rather than denaturing conditions

  • Consider using magnetic beads with different properties for separation

  • Validate recovery efficiency at each step with known standards

• Multiplexed Western blotting:

  • Use differentially labeled secondary antibodies for simultaneous detection

  • Strip and reprobe membranes sequentially with documentation between rounds

  • Consider size differences to avoid signal overlap

  • Use dual-color detection systems with appropriate controls

These strategies enable complex experimental designs investigating interactions between multiple tagged proteins within the same experimental system.

What are the most common causes of false positives/negatives with Myc tag antibodies and how can they be addressed?

Accurate interpretation of experimental results requires awareness of potential artifacts:

Implementing rigorous controls and validation steps significantly improves data reliability and reproducibility.

How can researchers validate the specificity of a Myc tag antibody in their experimental system?

Thorough validation ensures experimental reliability:

• Essential controls:

  • Non-transfected/non-transformed negative controls

  • Cells expressing known Myc-tagged proteins as positive controls

  • Competitive inhibition with excess Myc peptide (EQKLISEEDL)

  • Secondary antibody-only controls to assess non-specific binding

• Cross-validation approaches:

  • Compare detection with multiple anti-Myc antibody clones

  • Validate with alternative detection methods (e.g., fluorescent proteins)

  • Use alternative tagging systems (FLAG, HA) on the same protein

  • Perform mass spectrometry analysis of immunoprecipitated material

• Specificity testing:

  • Examine size-appropriate bands on Western blots

  • Perform siRNA/shRNA knockdown of tagged construct to confirm signal reduction

  • Use CRISPR/Cas9 to tag endogenous proteins for physiological expression level controls

  • Compare subcellular localization with known distribution patterns or GFP fusion proteins

• Advanced validation:

  • Use proximity ligation assays to verify co-localization specificity

  • Employ super-resolution microscopy to assess spatial distribution

  • Perform epitope mapping to confirm precise antibody binding sites

  • Conduct functional rescue experiments to verify biological activity of tagged proteins

Thorough validation increases confidence in experimental outcomes and facilitates meaningful interpretation of results.

How are new technological developments enhancing Myc tag detection and applications?

Recent technological advances are expanding the utility of Myc-tagged proteins:

• Single-molecule detection:

  • Super-resolution microscopy enables visualization of individual Myc-tagged proteins

  • Single-molecule pull-down techniques allow analysis of protein complexes at endogenous levels

  • Nanobody-based detection improves spatial resolution and penetration

  • Improved signal amplification methods enable detection of low-abundance proteins

• Live-cell applications:

  • Split-tag complementation systems for studying protein-protein interactions

  • FRET/BRET-based approaches for real-time interaction monitoring

  • Development of cell-permeable anti-Myc antibody fragments

  • Integration with optogenetic tools for spatiotemporal control

• High-throughput methodologies:

  • Microfluidic antibody validation platforms for systematic comparison

  • Automated image analysis algorithms for quantitative assessment

  • Barcode-enabled multiplexing for simultaneous detection of multiple Myc-tagged proteins

  • Integration with single-cell technologies for heterogeneity analysis

• Next-generation antibodies:

  • Recombinant antibody technologies improving batch-to-batch consistency

  • Engineering of high-affinity variants with reduced context sensitivity

  • Development of bispecific antibodies for multiplexed detection

  • Nanobody and single-domain antibody alternatives with improved tissue penetration

These advances are expanding the experimental possibilities for researchers using Myc-tagged proteins and improving data quality and reproducibility.

What considerations are important when integrating Myc tag detection with other advanced research methodologies?

As research techniques evolve, integrating Myc tag detection with advanced methodologies requires careful consideration:

• Integration with CRISPR/Cas9 genome editing:

  • Design appropriate homology-directed repair templates for endogenous tagging

  • Consider tag position effects on protein function and antibody accessibility

  • Validate edited clones with multiple detection methods

  • Account for potential heterogeneity in edited populations

• Compatibility with advanced imaging techniques:

  • Optimize fixation and permeabilization protocols for super-resolution microscopy

  • Consider probe size limitations for techniques like STORM or PALM

  • Validate spatial resolution with known structural features

  • Implement appropriate controls for colocalization studies

• Adaptation for proteomics applications:

  • Optimize elution conditions to maintain complex integrity

  • Consider on-bead digestion protocols to improve peptide recovery

  • Implement isotopic labeling strategies for quantitative comparisons

  • Validate results with alternative purification strategies

• Single-cell analysis considerations:

  • Account for expression level heterogeneity in cell populations

  • Optimize fixation and permeabilization for intracellular epitope access

  • Implement appropriate compensation controls for multi-parameter analysis

  • Consider cell cycle effects on tagged protein expression and localization

Careful integration of Myc tag detection with these advanced methodologies enables sophisticated experimental designs while maintaining data reliability.