mycn Antibody

What is MYCN and why is it significant in cancer research?

MYCN is a proto-oncogene belonging to the Myc family that encodes a nuclear-localized bHLH transcription factor. The human MYCN protein has 464 amino acid residues with a protein mass of 49.6 kilodaltons . Unlike c-MYC which is expressed in many proliferating cells, MYCN expression is more restricted, with highest levels during embryonic development and in specific adult tissues like developing B-cells .

MYCN is particularly significant in neuroblastoma research, where gene amplification is strongly associated with rapid disease progression and poor prognosis . The protein functions in transcriptional regulation by binding to E-box sequences in DNA, facilitating expression of genes essential for cellular growth and metabolism . Recent research has also identified MYCN as an immunosuppressive oncogene that negatively regulates the expression of ligands for NK cell-activating receptors, contributing to tumor immune evasion .

How do I select the appropriate MYCN antibody for my experimental needs?

When selecting an MYCN antibody, consider these methodological factors:

• Application compatibility: Verify validation data for your specific application (WB, IHC, IF, ChIP, etc.)

• Species reactivity: Ensure compatibility with your experimental system (human, mouse, rat)

• Clonality:

  • Monoclonal antibodies (e.g., NCM II 100) offer higher specificity

  • Polyclonal antibodies may provide stronger signals by recognizing multiple epitopes

• Epitope location: Consider whether N-terminal, C-terminal, or internal epitopes are better for your application

• Format requirements: Determine if you need unconjugated antibody or conjugates (HRP, FITC, PE, Alexa Fluor®)

Review validation data including Western blot images showing the expected molecular weight band (approximately 50-70 kDa) and positive controls such as MYCN-amplified neuroblastoma cell lines .

What controls should be included when working with MYCN antibodies?

A rigorous experimental design requires appropriate controls:

• Positive controls:

  • MYCN-amplified neuroblastoma cell lines (IMR-32, SMS-KCNR, Kelly)

  • Transfected cells overexpressing MYCN

  • Tissues known to express MYCN (embryonic brain, neuroblastoma tissue)

• Negative controls:

  • Cell lines with low or undetectable MYCN expression

  • MYCN-silenced cells (siRNA or CRISPR/Cas9 knockout)

  • Isotype controls for non-specific binding

• Specificity controls:

  • Validation against other Myc family members to check cross-reactivity

  • Correlation with MYCN mRNA expression or gene amplification status

• Technical controls:

  • Secondary antibody-only controls to assess background

  • Internal loading controls for Western blots (β-actin, GAPDH)

How can I distinguish between MYCN and c-MYC detection with antibodies?

Distinguishing between MYCN and c-MYC is challenging due to their structural similarities:

• Antibody selection: Use monoclonal antibodies specifically validated to not cross-react with other Myc family members

• Expression patterns: MYCN and c-MYC expression are often inversely correlated in neuroblastoma cells, which can aid differentiation

• Molecular weight differentiation:

  • MYCN typically appears at 62-70 kDa on Western blots

  • c-MYC usually appears at 57-60 kDa

• Validation approaches:

  • Test antibodies on cell lines with differential expression of MYCN and c-MYC

  • Be cautious with polyclonal c-MYC antibodies, which may cross-react with MYCN in cells with high MYCN expression (as observed in SH-EP, IMR5/75, and Kelly cell lines)

  • Perform parallel experiments with specific c-MYC antibodies for comparison

• Functional validation: Use siRNA knockdown specific to either MYCN or c-MYC to confirm antibody specificity

What are optimal conditions for MYCN detection in different applications?

For optimal MYCN detection across applications:

Western Blotting:

• Recommended dilutions: 1:500-1:3000

• Sample preparation: Use fresh lysates with protease inhibitors to prevent degradation

• Blocking: 5% BSA or milk to reduce background

• Expected molecular weight: 50-70 kDa (observe that actual detected weight may be higher than predicted 50 kDa)

Immunohistochemistry:

• Dilution range: 1:50-1:500

• Antigen retrieval: Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for optimal results

• Detection systems: Consider signal amplification for low-expressing samples

• Counterstaining: Hematoxylin for nuclear contrast (as MYCN is primarily nuclear)

Immunofluorescence:

• Fixation: 4% paraformaldehyde preserves nuclear structure

• Permeabilization: Optimize with Triton X-100 concentration for nuclear access

• Blocking: Normal serum matching secondary antibody species

• Nuclear counterstain: DAPI or Hoechst

What experimental challenges are common with MYCN antibodies and how can they be addressed?

Common challenges include:

• Background/non-specific staining:

  • Solution: Optimize blocking conditions, increase washing steps, use monoclonal antibodies, perform absorption controls

• Variable sensitivity across applications:

  • Solution: Validate each antibody specifically for your application, as performance may vary between WB, IHC, and IF

• Discrepancies between mRNA expression, gene amplification, and protein detection:

  • Solution: Analyze multiple parameters simultaneously (FISH, qPCR, Western blot, IHC) to obtain comprehensive assessment

• Protein instability and rapid degradation:

  • Solution: Include proteasome inhibitors in lysates, maintain samples at cold temperatures, process rapidly

• Cross-reactivity with other Myc family members:

  • Solution: Use antibodies with validated specificity against c-MYC and L-MYC, include appropriate controls

How can MYCN antibodies be used to study protein stability as a prognostic indicator?

MYCN protein stability has emerged as a more reliable prognostic indicator than gene amplification alone . Methodological approaches include:

• Quantitative analysis methods:

  • Western blot with densitometry standardized to recombinant MYCN controls

  • IHC with digital image analysis and standardized scoring systems

  • Proteasome inhibition studies to assess degradation kinetics

• Clinical correlation approaches:

  • Compare MYCN protein levels with patient outcomes using standardized scoring

  • Correlate IHC results with FISH to identify discordant cases (amplified but not expressed, or expressed without amplification)

  • Multivariate analysis controlling for other prognostic factors

• Stability assessment:

  • Cycloheximide chase experiments to measure protein half-life

  • Analysis of post-translational modifications affecting stability

  • Co-immunoprecipitation to identify protein interactions influencing degradation

This approach addresses the critical finding that "tumors with MYCN amplification could not express protein" in some cases, while in others, "MYCN protein could be isolated from tumors without gene amplification" .

How can MYCN antibodies be used to investigate the role of MYCN in immune evasion?

Recent research has identified MYCN as an immunosuppressive oncogene . To study this function:

• Expression correlation studies:

  • Use flow cytometry to correlate MYCN expression with surface levels of NK cell-activating receptor ligands (MICA, ULBPs, PVR)

  • Perform regression analysis between MYCN levels and immune activation markers

• Functional assessments:

  • In MYCN-inducible cell lines (e.g., Tet-21/N), analyze changes in immune ligand expression following MYCN modulation

  • Conduct degranulation and cytotoxicity assays to assess NK cell-mediated killing of tumor cells with varying MYCN levels

• Mechanistic investigations:

  • ChIP studies to identify direct transcriptional regulation of immune genes by MYCN

  • Co-immunoprecipitation to detect protein interactions with immune signaling components

• Translational approaches:

  • Analyze primary neuroblastoma samples for correlations between MYCN expression and immune infiltration

  • Test MYCN inhibitors for their ability to enhance immunotherapy responses

Data from patient samples confirm "an inverse correlation between the expression of MYCN and that of ligands for NK-cell-activating receptors," suggesting MYCN expression could serve as a biomarker to "predict the efficacy of NK-cell-based immunotherapy in NB patients" .

How can ChIP experiments with MYCN antibodies illuminate transcriptional networks?

Chromatin Immunoprecipitation (ChIP) with MYCN antibodies enables genome-wide mapping of MYCN binding sites:

• Antibody selection considerations:

  • Use antibodies validated specifically for ChIP applications

  • Select antibodies recognizing epitopes not involved in DNA binding

  • Consider whether fixation protocols might obscure the epitope

• Experimental design:

  • Include input controls and IgG controls

  • Perform in multiple cell lines with varying MYCN expression levels

  • Consider sequential ChIP to distinguish MYCN from c-MYC binding sites

• Data analysis approaches:

  • Identify E-box motifs and other binding sequences

  • Integrate with transcriptomic data to correlate binding with expression

  • Compare binding patterns between MYCN-amplified and non-amplified samples

• Validation strategies:

  • Confirm selected binding sites with ChIP-qPCR

  • Perform reporter assays to verify functional significance

  • Use CRISPR-based approaches to mutate binding sites

These approaches have revealed that "distinct transcriptional MYCN/c-MYC activities are associated with different neuroblastoma subtypes," providing insights into disease mechanisms .

How does MYCN protein detection compare to gene amplification testing in clinical settings?

Comparing protein detection with gene amplification reveals important clinical insights:

• Concordance analysis:

  • Studies show variable concordance rates between FISH and IHC (as low as 36.4% in some cohorts)

  • Some tumors with MYCN amplification do not express detectable protein

  • Some non-amplified tumors express significant MYCN protein levels

• Prognostic value comparison:

  • Protein stability has emerged as "a better prognostic indicator" than gene amplification alone

  • Combined analysis of both parameters improves risk stratification

  • Protein expression may better reflect actual MYCN oncogenic activity

• Methodological considerations:

  • Standardized scoring systems for IHC are needed for consistent assessment

  • Automated image analysis can reduce interpreter variation

  • Multiple antibody validation improves reliability of protein detection

• Implementation approaches:

  • Consider routine parallel testing of both FISH and IHC in clinical samples

  • Develop composite scoring systems incorporating both gene and protein status

  • Serial monitoring of protein levels during treatment may provide additional prognostic information

What methodological approaches can detect post-translational modifications of MYCN?

Post-translational modifications (PTMs) significantly impact MYCN stability and function:

• Phosphorylation detection:

  • Use phospho-specific antibodies targeting known modification sites

  • Perform lambda phosphatase treatment as controls

  • Combine with proteasome inhibitors to capture unstable phosphorylated forms

• Ubiquitination analysis:

  • Immunoprecipitate MYCN under denaturing conditions

  • Probe with anti-ubiquitin antibodies

  • Use deubiquitinating enzyme inhibitors during sample preparation

• Stability relationship studies:

  • Correlate PTM status with protein half-life using cycloheximide chase

  • Compare PTM patterns in MYCN-amplified versus non-amplified samples

  • Investigate kinase inhibitors that may modulate MYCN stability

• Functional significance assessment:

  • Create point mutations at modification sites

  • Analyze impact on protein stability, localization, and transcriptional activity

  • Correlate modification status with treatment response

Understanding PTMs provides mechanistic insights into why "MYCN protein could be isolated from tumors without gene amplification" and why some amplified tumors may not express the protein .

How can MYCN detection be optimized for clinical diagnostics and patient stratification?

For clinical implementation, standardization is essential:

• IHC optimization:

  • Establish consensus protocols for fixation, processing, and staining

  • Use automated staining platforms for consistency

  • Implement digital image analysis with validated scoring algorithms

  • Include calibration controls on each slide

• Combined testing strategies:

  • Integrate FISH, RT-qPCR, and IHC results

  • Develop algorithms weighing the relative contributions of each method

  • Include assessment of MYCN-regulated genes as functional readouts

• Quality assurance measures:

  • Participate in inter-laboratory proficiency testing

  • Use standardized positive and negative controls

  • Implement regular antibody validation

  • Document lot-to-lot variability

• Result interpretation guidelines:

  • Establish clear reporting formats

  • Define clinically relevant cutoffs

  • Include reliability metrics with results

  • Provide guidance for discordant results between methods

These approaches address the finding that protein detection "sensitivity and specificity were substandard" with earlier antibodies , offering improved diagnostic accuracy for patient stratification.

How might MYCN antibodies contribute to developing targeted therapeutics?

MYCN antibodies are instrumental in therapeutic development:

• Target validation approaches:

  • Use antibodies to confirm MYCN expression in preclinical models

  • Assess correlation between MYCN levels and drug sensitivity

  • Monitor MYCN degradation in response to indirect targeting strategies

• Screening platforms:

  • Develop high-throughput assays using MYCN antibodies to identify compounds inducing degradation

  • Create reporter systems with epitope-tagged MYCN for live monitoring

  • Implement proximity-based assays to screen for disruptors of key protein interactions

• Therapeutic antibody development:

  • While direct targeting with antibodies is challenging due to nuclear localization

  • Antibody-drug conjugates could target MYCN-driven surface markers

  • Intrabodies (intracellular antibodies) might be delivered via newer technologies

• Treatment response monitoring:

  • Use MYCN antibodies to assess pharmacodynamic response to targeted therapies

  • Analyze changes in MYCN-regulated pathways following treatment

  • Identify resistance mechanisms by comparing pre- and post-treatment MYCN status

These approaches could help overcome the current challenges in directly targeting transcription factors like MYCN.

What technological advances are improving MYCN antibody performance and applications?

Emerging technologies are enhancing MYCN antibody applications:

• Single-cell analysis methods:

  • Imaging mass cytometry for multiplexed protein detection in tissues

  • Single-cell Western blotting for heterogeneity assessment

  • Proximity ligation assays for protein interaction studies at single-cell resolution

• Next-generation sequencing integration:

  • CUT&RUN as an alternative to traditional ChIP with lower input requirements

  • CITE-seq for simultaneous surface marker and transcriptome analysis

  • Spatial transcriptomics correlated with MYCN protein localization

• Antibody engineering advances:

  • Recombinant antibody production for improved consistency

  • Nanobodies with enhanced nuclear penetration for live-cell imaging

  • Site-specific conjugation methods for more homogeneous antibody reagents

• Computational approaches:

  • Machine learning algorithms for automated scoring of IHC

  • Systems biology integration of MYCN protein networks

  • Predictive modeling of MYCN stability based on post-translational modifications

These technological advances address historical challenges with antibody "specificity and sensitivity" that were "substandard" with earlier reagents .