c-MYC Monoclonal Antibody

What is c-MYC protein and why is it essential in cellular research?

The c-MYC protein functions as a transcription factor encoded by the c-MYC gene located on human chromosome 8q24. As a key regulator of numerous cellular processes, c-MYC influences cell proliferation, differentiation, apoptosis, and cell cycle progression. Its significance in research stems from its frequent dysregulation in various diseases, particularly cancer, making it a critical target for therapeutic intervention .

The protein's role in vasculogenesis and angiogenesis has drawn considerable attention in cancer research, as these processes are essential for tumor development. Understanding c-MYC function requires sensitive and specific detection methods, with monoclonal antibodies serving as primary tools for quantifying expression levels and visualizing cellular localization .

How do various c-MYC monoclonal antibodies differ in specificity and application?

Different c-MYC monoclonal antibodies exhibit significant variation in their detection capabilities and context-dependent sensitivities. The following table compares key c-MYC antibody clones based on published research:

The 9E10 clone, despite being the most widely cited in literature, demonstrates significant context-dependent detection variability compared to newer antibodies like 4A6 and 9B11, which show much more uniform reactivity across diverse experimental conditions . Importantly, while 9E10 was previously thought to recognize only human c-MYC, research has demonstrated its cross-reactivity with murine c-MYC, particularly in denatured conditions .

What are the methodological considerations for Western blot detection of c-MYC?

Successful Western blot detection of c-MYC requires careful optimization of several parameters. Based on empirical evidence, the following methodology yields reliable results:

• Sample Preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors if detecting phosphorylated forms.

• Protein Loading: For endogenous c-MYC detection, load 20-50 μg of total protein per lane. For tagged proteins, 10-20 μg is typically sufficient.

• Antibody Concentration: The optimal working concentration varies by antibody clone. For example, 9E10 (MAB3696) has been effectively used at 2 μg/mL for HeLa and Daudi cell lysates .

• Detection System: Use HRP-conjugated secondary antibodies with appropriate sensitivity. Modern antibodies like THE™ c-Myc Tag Antibody show excellent performance even at high dilutions (1:1000, equivalent to 1.0 μg/ml) .

• Expected Band Size: Endogenous c-MYC typically appears at approximately 52-74 kDa. Tagged proteins will show size shifts according to the fusion partner size .

Multiple scientific sources confirm that reducing conditions and appropriate buffer systems (e.g., Immunoblot Buffer Group 1) consistently improve detection quality .

How can researchers address context-dependent variability in c-MYC epitope tag recognition?

Context-dependent variability in c-MYC epitope tag recognition presents a significant challenge for researchers. Studies have demonstrated that epitope tag position and neighboring sequences substantially affect antibody recognition, with implications for data interpretation .

To mitigate this variability:

• Antibody Selection: Choose purpose-made monoclonal antibodies (like 4A6 or 9B11) that demonstrate lower context sensitivity than legacy antibodies (particularly 9E10) .

• Multiple Antibody Validation: Employ at least two different antibody clones targeting different epitopes to confirm results, especially in novel experimental systems.

• Peptide Microarray Analysis: When working with a new experimental system, consider peptide microarray analysis to evaluate context sensitivity for your specific application. This approach has been instrumental in revealing the high context sensitivity of 9E10 .

• Tag Positioning: When designing tagged constructs, place the tag at both N-terminal and C-terminal positions in parallel experiments to confirm expression patterns, as antibody detection can be significantly affected by tag position .

• Control Selection: Include both positive and negative controls with known tag accessibility profiles.

This strategic approach has been validated through multiple studies showing that newer, purpose-made monoclonal antibodies provide more consistent detection across experimental contexts .

How do phosphorylation states affect c-MYC detection and function?

The phosphorylation state of c-MYC significantly impacts both its detection and biological function. Research has established important relationships between specific phosphorylation sites and cellular processes:

• Key Phosphorylation Sites: Phosphorylation at Thr58/Ser62 by glycogen synthase kinase 3, cyclin-dependent kinase, ERK2, and C-Jun N-terminal Kinase (JNK) has been functionally associated with cell proliferation and cell cycle regulation .

• Detection Methodologies: When studying phosphorylated c-MYC:

  • Use phospho-specific antibodies in conjunction with standard c-MYC antibodies

  • Include phosphatase inhibitors in lysis buffers

  • Validate with phosphatase treatment controls

  • Consider lambda phosphatase treatment of parallel samples to confirm specificity

• Functional Implications: Phosphorylation state directly influences c-MYC protein stability, transcriptional activity, and protein-protein interactions. For example, Thr58 phosphorylation typically promotes c-MYC degradation, while Ser62 phosphorylation can stabilize the protein.

• Cancer-Specific Alterations: Many tumors exhibit dysregulated c-MYC phosphorylation patterns that contribute to protein stabilization and oncogenic activity .

For accurate phosphorylation analysis, researchers should implement rapid sample processing techniques to preserve native phosphorylation states and consider using quantitative methods such as Phos-tag gels to separate different phosphorylated forms.

What strategies resolve cross-reactivity issues between human and murine c-MYC detection?

Cross-reactivity between human and murine c-MYC presents significant challenges for researchers working with mixed-species samples. Structural analysis reveals that the C-terminal regions of human and murine c-MYC share a highly conserved sequence (amino acids 418-439) with only one terminal exchange (C438G), while the pre-C-terminal region (amino acids 408-417) contains four amino acid differences .

To address cross-reactivity issues:

• Antibody Selection Based on Epitope Recognition:

  • The 9E10 antibody, raised against human c-MYC C-terminal amino acids 408-439, was historically thought to be human-specific but has been demonstrated to cross-react with denatured murine c-MYC .

  • Choose antibodies with validated species specificity for your experimental system.

• Competitive Peptide Blocking:

  • Implement peptide competition assays to confirm specificity. Research has shown that immunostaining with both 9E10 and polyclonal antibodies in human and murine cell lines can be competed by peptides corresponding to murine pre-C-terminal amino acids 408-417 .

• Multiple Detection Methods:

  • Confirm results using multiple techniques (Western blot, immunofluorescence, etc.) as cross-reactivity can vary with the method used.

  • Include controls from both species in parallel experiments.

• Genetic Approaches:

  • In cases requiring absolute specificity, consider genetic approaches such as CRISPR/Cas9 knockout controls or species-specific siRNA knockdown to validate antibody specificity.

These strategies have been empirically validated through comparative studies using human (SV80) and murine (L929) cell lines, demonstrating that what was initially considered human-specific reactivity often extends to murine samples in both Western blot and immunofluorescence applications .

How can immunofluorescence protocols be optimized for c-MYC detection?

Optimizing immunofluorescence (IF) protocols for c-MYC detection requires careful consideration of multiple parameters to ensure specific signal and low background. Based on published methodologies:

• Fixation Method Selection:

  • For nuclear c-MYC detection: 4% paraformaldehyde (10-15 minutes) followed by 0.2% Triton X-100 permeabilization yields optimal nuclear signal preservation.

  • For detecting both nuclear and cytoplasmic c-MYC: Methanol fixation (-20°C, 10 minutes) provides good results while preserving epitope accessibility.

• Antibody Selection and Concentration:

  • THE™ Anti-c-Myc-tag Monoclonal Antibody (Mouse) has demonstrated excellent results in IF applications for c-Myc tagged proteins .

  • Starting dilution of 1:200-1:500 is recommended, with optimization for each experimental system.

• Signal Amplification Strategies:

  • For low-abundance targets: Consider tyramide signal amplification methods.

  • For multiplex applications: Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity.

• Background Reduction:

  • Extended blocking (1-2 hours) with 5% BSA and 5% normal serum from the secondary antibody host species.

  • Include 0.1-0.3% Triton X-100 in blocking and antibody dilution buffers to reduce non-specific binding.

• Controls:

  • Include c-MYC-negative cells or tissues as negative controls.

  • For tagged proteins, untransfected cells serve as essential controls.

  • Consider peptide competition controls for validation.

Implementation of these methodological refinements has been demonstrated in successful visualization of c-MYC in 293 cells transfected with c-MYC tag protein using THE™ Anti-c-Myc-tag Monoclonal Antibody . This approach maximizes signal-to-noise ratio while preserving biological relevance.

What validation strategies confirm antibody specificity in c-MYC research?

Rigorous validation of c-MYC antibody specificity is essential for reliable research outcomes. Comprehensive validation strategies include:

• Knockout/Knockdown Controls:

  • Knockout validation, such as with the c-Myc Rabbit Monoclonal Antibody (CAB19032), represents the gold standard for specificity confirmation .

  • siRNA or shRNA knockdown provides an accessible alternative when knockout models are unavailable.

• Multiple Antibody Concordance:

  • Compare results using antibodies targeting different epitopes of c-MYC.

  • Concordant results across antibodies significantly increase confidence in specificity.

• Peptide Competition Assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific staining.

  • Include non-relevant peptides as negative controls.

• Recombinant Expression Systems:

  • Overexpression of tagged c-MYC in cell lines with low endogenous expression.

  • Demonstration of signal increase proportional to expression level.

• Cross-Species Reactivity Testing:

  • Test antibodies against samples from multiple species to map cross-reactivity.

  • Document unexpected cross-reactivity, such as that observed with 9E10 antibody against murine c-MYC .

• Application-Specific Validation:

  • Validate separately for each application (Western blot, immunofluorescence, immunohistochemistry).

  • Different applications expose different epitopes and may affect specificity.

These validation approaches provide complementary evidence for antibody specificity, with knockout validation offering the most definitive proof of specificity, as demonstrated in recent antibody development protocols .

What are the critical parameters for quantifying c-MYC expression accurately?

Accurate quantification of c-MYC expression requires meticulous attention to technical parameters and appropriate normalization strategies:

• Sample Preparation Consistency:

  • Standardize cell harvesting protocols to minimize variation in c-MYC levels due to cell cycle effects.

  • Process samples rapidly due to the short half-life of c-MYC protein (approximately 20-30 minutes).

• Western Blot Quantification:

  • Use gradient gels (4-12% or 4-20%) to maximize resolution.

  • Implement standard curves with recombinant c-MYC for absolute quantification.

  • Employ fluorescent secondary antibodies for wider linear dynamic range compared to chemiluminescence.

  • Multiple loading amounts demonstrate detection linearity, as shown with THE™ c-Myc Tag Antibody .

• Image Analysis Parameters:

  • Use software that permits background subtraction.

  • Define regions of interest consistently across samples.

  • Implement blind analysis to prevent unconscious bias.

• Normalization Strategies:

  • For Western blot: Normalize to multiple housekeeping proteins rather than relying on a single reference protein.

  • For immunohistochemistry: Use automated image analysis with cell counting and intensity measurement.

  • For flow cytometry: Include fluorescence minus one (FMO) controls for accurate gating.

• Statistical Approaches:

  • Calculate coefficient of variation across technical replicates (should be <15%).

  • Implement ANOVA with post-hoc tests for multiple comparison conditions.

  • Report confidence intervals alongside means.

These methodological considerations have been validated across multiple studies and enable researchers to detect subtle but biologically significant changes in c-MYC expression levels with confidence and reproducibility.

How can researchers resolve non-specific binding in c-MYC Western blots?

Non-specific binding presents a common challenge in c-MYC Western blotting. Systematic troubleshooting strategies include:

• Antibody Selection and Optimization:

  • Newer purpose-made antibodies like 4A6 and 9B11 demonstrate significantly less non-specific binding than legacy antibodies like 9E10 .

  • Titrate antibody concentration; for example, THE™ c-Myc Tag Antibody has been shown to work efficiently at high dilutions (1:1000) .

• Blocking Optimization:

  • For membrane-bound proteins: 5% non-fat dry milk in TBST.

  • For phosphoproteins: 5% BSA in TBST.

  • Extended blocking times (2+ hours) often reduce non-specific binding.

• Washing Protocol Enhancement:

  • Increase wash duration and volume.

  • Add 0.05% SDS to wash buffer for highly hydrophobic proteins.

  • Implement programmed washing platforms for consistent agitation.

• Sample Preparation Modifications:

  • Pre-clear lysates with Protein A/G beads to remove proteins that bind non-specifically.

  • Include detergent optimization steps (RIPA vs. NP-40 vs. Triton X-100).

  • Use freshly prepared samples to minimize degradation products.

• Secondary Antibody Considerations:

  • Use highly cross-adsorbed secondary antibodies.

  • Consider secondary antibody-only controls to identify direct non-specific binding.

• Detection System Adjustment:

  • For chemiluminescence: Shorter exposure times often reduce background.

  • For fluorescent detection: Optimize scanner settings and use appropriate filters.

Implementation of these strategies has been shown to significantly improve signal-to-noise ratio in Western blot analysis of c-MYC. Studies comparing antibody performance demonstrate that methodical optimization can transform uninterpretable blots into clear, quantifiable results .

What factors influence variability in c-MYC detection across experimental replicates?

Experimental variability in c-MYC detection stems from multiple sources that must be systematically addressed:

• Biological Variability Factors:

  • Cell cycle stage: c-MYC levels fluctuate significantly throughout the cell cycle; synchronization methods can reduce this variability.

  • Cell density effects: Contact inhibition alters c-MYC expression; standardize seeding density and harvest at consistent confluence.

  • Serum factors: Growth factors in serum induce c-MYC; use consistent serum lots or switch to defined media for sensitive experiments.

• Technical Variability Sources:

  • Antibody lot-to-lot variation: Qualify new lots against reference standards.

  • Detection system consistency: Calibrate imaging systems regularly.

  • Protein extraction efficiency: Standardize lysis conditions and protein quantification methods.

• Experimental Design Approaches:

  • Include biological and technical replicates in experimental design.

  • Implement randomization of sample processing order.

  • Consider blocking experimental factors that cannot be controlled.

• Analytical Considerations:

  • Use appropriate statistical tests for variability assessment.

  • Apply outlier identification methods judiciously.

  • Consider power analysis to determine appropriate sample sizes.

• Epitope Accessibility Variations:

  • Context-dependent accessibility: As demonstrated for 9E10 antibody, epitope recognition can vary dramatically based on tag position and neighboring sequences .

  • Establish consistent protein denaturation conditions for Western blotting.

Data from peptide microarray analyses confirm that antibodies like 9E10 display high context-dependent detection variability, while others such as 4A6 and 9B11 show much more uniform reactivity . These findings emphasize the importance of antibody selection as a primary factor in managing experimental variability.

How can contradictory results between different c-MYC antibodies be reconciled?

Contradictory results between different c-MYC antibodies occur frequently and require systematic analytical approaches for resolution:

• Epitope Mapping Analysis:

  • Different antibodies recognize distinct epitopes that may be differentially accessible in various experimental contexts.

  • Compare epitope locations with known post-translational modification sites that might mask recognition.

  • The 9E10 antibody, for example, recognizes C-terminal amino acids 408-439 of human c-MYC, which may be affected by structural changes .

• Experimental Validation Hierarchy:

  • Establish a validation hierarchy based on complementary techniques.

  • Prioritize results from knockout-validated antibodies over non-validated antibodies .

  • Consider orthogonal approaches like mass spectrometry to resolve persistent contradictions.

• Context-Dependent Detection Assessment:

  • Evaluate whether contradictions are related to context-dependent detection variability.

  • Test antibodies under identical conditions using recombinant proteins with tags in different positions.

  • Studies have shown that antibodies like 9E10 display high context-dependent variability, while others (4A6, 9B11) show more consistent detection .

• Cross-Reactivity Investigation:

  • Determine if contradictions stem from unexpected cross-reactivity.

  • Include species-specific controls, as some antibodies (like 9E10) show cross-reactivity with murine c-MYC despite being marketed as human-specific .

• Technical Optimization:

  • Systematically vary experimental conditions (fixation methods, blocking agents, etc.).

  • Document conditions under which concordance between antibodies is achieved.

• Literature Integration:

  • Compare findings with published literature reporting similar contradictions.

  • Consider unpublished technical challenges that may be shared through research communities.

This structured approach has successfully resolved contradictions in c-MYC detection across multiple experimental systems, as documented in comparative antibody studies .

What are the emerging trends in c-MYC antibody development and application?

Recent advances in c-MYC antibody technology are transforming research capabilities through several key developments:

• Knockout-Validated Antibody Production:

  • Next-generation antibodies, such as the KO Validated c-Myc Rabbit Monoclonal Antibody (CAB19032), represent a significant advance in specificity validation .

  • This validation approach substantially reduces false positive results and increases confidence in experimental findings.

• Reduced Context Sensitivity:

  • Purpose-made monoclonal antibodies with much less context sensitivity than legacy antibodies are becoming standard.

  • Comparative studies demonstrate that newer antibodies like 4A6 and 9B11 provide more uniform reactivity across diverse experimental conditions than the widely used 9E10 antibody .

• Multi-Application Optimization:

  • Modern antibodies are increasingly designed and validated for multiple applications rather than single-use scenarios.

  • THE™ c-Myc Tag Antibody exemplifies this trend, with demonstrated efficacy in Western blot, immunoprecipitation, and immunofluorescence applications .

• Integration with Advanced Detection Systems:

  • Compatibility with cutting-edge technologies like Simple Western™ automated Western blotting systems enhances reproducibility and quantification accuracy .

  • Adaptation to multiplex detection platforms enables simultaneous analysis of c-MYC alongside interaction partners or pathway components.

• Therapeutic Development Connection:

  • Antibodies developed for research applications are increasingly informing therapeutic antibody development targeting c-MYC in cancer.

  • Bifunctional antibodies coupling c-MYC recognition with effector recruitment represent an emerging research frontier.

These advancements collectively enhance the precision, reliability, and utility of c-MYC detection in both basic research and translational applications, with direct implications for understanding c-MYC's role in disease progression and developing targeted interventions.

What key considerations should inform experimental design for c-MYC research?

Strategic experimental design for c-MYC research should incorporate these critical considerations:

• Antibody Selection Strategy:

  • Choose antibodies based on validated specificity and context sensitivity profiles rather than citation frequency.

  • Consider the specific experimental context; for example, antibodies demonstrating excellent performance in Western blot may not perform equally well in immunohistochemistry .

• Biological Relevance Planning:

  • Account for c-MYC's short half-life and rapid turnover in experimental timing.

  • Consider cell cycle effects on c-MYC expression levels when designing experiments and controls.

  • Include physiologically relevant models alongside established cell lines.

• Comprehensive Controls Framework:

  • Implement knockout/knockdown controls whenever possible to validate specificity.

  • Include both positive controls (known c-MYC expression) and negative controls (c-MYC-negative or depleted samples).

  • For tagged constructs, include both N-terminal and C-terminal tag positions to address potential context-dependent detection issues .

• Cross-Method Validation:

  • Confirm key findings using complementary detection methods.

  • Consider quantitative PCR to correlate protein levels with transcriptional activity.

  • Implement functional assays to connect expression patterns with biological outcomes.

• Data Integration Approach:

  • Design experiments with bioinformatic integration in mind.

  • Consider how experimental data will be normalized and compared across different platforms.

  • Plan for appropriate statistical analyses based on anticipated data distribution.