c-myc Antibody

What is c-myc and why is it significant in cellular research?

c-myc is a 454-amino acid residue protein that functions as a transcription factor encoded by the MYC gene in humans. It binds DNA both non-specifically and specifically recognizes the core sequence 5'-CAC[GA]TG-3'. Located primarily in the nucleus, c-myc undergoes several post-translational modifications (glycosylation, ubiquitination, and phosphorylation) that regulate its function and stability. This protein plays crucial roles in cell cycle progression, apoptosis, and cellular transformation by regulating transcription of specific target genes .

The significance of c-myc in research stems from its widespread expression across tissue types and its involvement in numerous cellular processes. Mutations, overexpression, rearrangement, and translocation of MYC have been associated with various hematopoietic tumors, leukemias, and lymphomas, including Burkitt lymphoma (BL) . Additionally, c-myc has been identified in specific cell populations including Early Erythroid Cells and Naive Thymus-Derived CD8+ αβ T Cells .

What criteria should researchers consider when selecting a c-myc antibody?

When selecting a c-myc antibody, researchers should evaluate several critical factors:

• Validated applications: Verify the antibody has been validated for your specific application (WB, IHC, ICC, IP, ChIP, etc.) with published data or manufacturer validation .

• Epitope specificity: Consider which region of c-myc the antibody recognizes. Some antibodies target specific domains or post-translational modifications .

• Species reactivity: Ensure compatibility with your experimental model. Available antibodies show varying reactivity patterns across species (human, mouse, rat, etc.) .

• Clonality: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals but with potential for more cross-reactivity .

• Format requirements: Determine if your experiment requires unconjugated antibodies or specific conjugates (PE, Alexa Fluor, biotin, etc.) .

• Validation methods: Review how the antibody was validated. High-quality antibodies are often verified using knockout cell lines, overexpression systems, and multiple detection methods .

• Publication record: Consider antibodies with established track records in peer-reviewed literature, indicated by citation numbers in product listings .

How can researchers validate the specificity of a c-myc antibody?

Validating antibody specificity is essential for ensuring reliable experimental results. For c-myc antibodies, implement these validation strategies:

• Knockout/knockdown controls: Compare antibody reactivity between wild-type cells and c-myc knockout/knockdown cells. The search results show this approach: "Western blot shows lysates of HEK293T human embryonic kidney parental cell line and c-Myc knockout HEK293T cell line (KO)... A specific band was detected for c-Myc at approximately 62 kDa in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line" .

• Overexpression systems: Test antibody in cells overexpressing c-myc to confirm proportional signal increase.

• Multiple antibody comparison: Use several antibodies targeting different c-myc epitopes and compare staining patterns—consistent results across antibodies suggest specificity.

• Peptide competition assays: Pre-incubate the antibody with immunizing peptide before application—a specific antibody's signal should be significantly reduced.

• Cross-validation with different techniques: Confirm findings using orthogonal methods like mass spectrometry or RNA expression analysis.

• Positive and negative control samples: Include tissues or cell lines with known high and low c-myc expression levels as biological controls .

• Isotype controls: For flow cytometry and IHC applications, include appropriate isotype controls to establish background staining levels.

What are the optimal conditions for Western blot detection of c-myc?

Western blot detection of c-myc requires specific optimization due to its molecular weight characteristics and expression level variations:

Though the theoretical molecular weight of c-myc is ~49 kDa, it typically appears at 57-62 kDa on Western blots due to post-translational modifications . Researchers should be aware that c-myc detection is verified in multiple cell lines including "HeLa human cervical epithelial carcinoma cell line, HT-29 human colon adenocarcinoma cell line, Jurkat human acute T cell leukemia cell line, and LNCaP human prostate cancer cell line" .

When troubleshooting, remember that alternative translation initiations can produce isoforms with distinct N-termini, potentially resulting in additional bands .

What are the key considerations for immunofluorescence detection of c-myc?

Successfully detecting c-myc via immunofluorescence requires optimization of several parameters:

• Fixation method: Paraformaldehyde (4%) works well for preserving c-myc epitopes while maintaining cellular architecture. The research data shows successful detection in "immersion fixed HeLa human cervical epithelial carcinoma cell line" .

• Permeabilization: Thorough nuclear membrane permeabilization is essential since c-myc is primarily nuclear. Triton X-100 (0.1-0.5%) for 5-10 minutes post-fixation is usually effective.

• Antibody concentration: The search results indicate successful staining using "Rabbit Anti-Human c-Myc Monoclonal Antibody at 3 μg/mL" , which provides a good starting point for optimization.

• Incubation conditions: Optimal results were achieved with "3 hours at room temperature" , though overnight incubation at 4°C may improve signal in low-expressing samples.

• Detection system: Secondary antibodies with bright fluorophores enhance detection sensitivity. The example used "NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody" .

• Nuclear counterstain: DAPI counterstaining confirms nuclear localization of c-myc and helps with morphological assessment.

• Controls: Include positive control cell lines with known c-myc expression and negative controls (secondary antibody only, isotype control).

• Expected pattern: "Specific staining was localized to nuclei" , consistent with c-myc's known localization. Any significant cytoplasmic staining should be validated carefully.

How should researchers approach chromatin immunoprecipitation (ChIP) with c-myc antibodies?

Chromatin immunoprecipitation with c-myc antibodies requires careful planning and rigorous controls:

• Antibody selection: Choose antibodies specifically validated for ChIP applications . Based on the search results, several c-myc antibodies are specifically validated for ChIP assays.

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes works for most applications, but optimization may be needed for specific cell types.

• Sonication conditions: Aim for chromatin fragments between 200-500 bp. Verify fragmentation efficiency by gel electrophoresis before proceeding.

• Immunoprecipitation conditions:

  • Use 2-5 μg of antibody per IP reaction

  • Include 5-10% input control for normalization

  • Run parallel IgG isotype control to establish background levels

  • Consider pre-clearing lysates to reduce non-specific binding

• Washing stringency: Use progressively more stringent washing buffers to remove non-specific interactions while preserving specific c-myc-DNA complexes.

• Analysis approach:

  • qPCR for known targets (e.g., nucleolin promoter)

  • ChIP-seq for genome-wide binding analysis

  • Report data as percent input or fold enrichment over IgG

• Validation strategy: Confirm key findings with:

  • Alternative c-myc antibodies

  • Biological replicates

  • Functional validation of binding through expression analysis

Since c-myc "binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3'" , ensure your analysis accounts for both specific motif-directed binding and broader genomic associations.

What methodological approaches can distinguish between c-myc isoforms?

Detecting different c-myc isoforms resulting from alternative translation requires specialized approaches:

• High-resolution Western blotting:

  • Use 12-15% polyacrylamide gels or gradient gels (4-20%)

  • Extend running time to maximize separation of similar-sized proteins

  • Consider Phos-tag™ gels to separate phosphorylated isoforms

• Isoform-specific antibodies:

  • Select antibodies targeting N-terminal regions that differ between isoforms

  • Epitope mapping can confirm which isoforms are recognized

  • The search results note that "alternative translation initiations from an upstream, in-frame non-AUG (CUG) and a downstream AUG start site result in the production of two isoforms with distinct N-termini"

• Complementary techniques:

  • Mass spectrometry to identify isoform-specific peptides

  • Polysome profiling to analyze translation initiation sites

  • RT-PCR with primers targeting alternative start regions

• Functional validation:

  • Create constructs with mutated alternative start sites

  • Generate isoform-specific knockdown models

  • The search results highlight biological significance: "The synthesis of non-AUG initiated protein is suppressed in Burkitt's lymphomas, suggesting its importance in the normal function of MYC"

• Data presentation:

  • Clearly label molecular weight markers

  • Include positive controls expressing specific isoforms

  • Quantify relative abundance of different isoforms

This methodological approach is particularly important for c-myc research as isoform expression patterns may have functional consequences in normal physiology and disease states.

How can phospho-specific c-myc antibodies enhance understanding of c-myc regulation?

Phospho-specific c-myc antibodies provide crucial insights into c-myc regulation and function beyond what general c-myc antibodies offer:

• Biological significance of c-myc phosphorylation:

  • Phosphorylation at serine 62 (S62) stabilizes c-myc protein and enhances its transcriptional activity

  • Threonine 58 (T58) phosphorylation often promotes degradation

  • These modifications regulate c-myc half-life and activity in response to cellular signaling

• Methodological advantages:

  • Monitor specific activation states in response to signaling events

  • Correlate post-translational modifications with functional outcomes

  • Distinguish between total protein levels and active fractions

• Technical requirements for phospho-antibodies:

  • Sample preparation must include phosphatase inhibitors

  • Rapid processing to prevent dephosphorylation

  • May require specialized fixation protocols for IHC/IF applications

• Experimental design considerations:

  • Always run parallel detection of total c-myc for normalization

  • Include appropriate controls (phosphatase-treated samples)

  • Consider temporal dynamics of phosphorylation events

The search results indicate the availability of phospho-specific antibodies: "Anti-c-Myc (phospho S62) antibody... Citations: (38)" , suggesting substantial research interest in this approach. These antibodies are particularly valuable for studying how c-myc responds to growth factor signaling, stress conditions, and therapeutic interventions that may alter its phosphorylation state.

What approaches can integrate c-myc protein detection with functional readouts?

Integrating c-myc protein detection with functional assessments provides deeper insights into its biological roles:

• Multiplexed flow cytometry approaches:

  • Combine c-myc antibodies with markers of proliferation (Ki-67, BrdU incorporation)

  • Include apoptosis indicators (Annexin V, cleaved caspase-3)

  • Add cell cycle phase markers (cyclins, DNA content)

  • Correlate c-myc levels with stemness markers in relevant systems

• Multiparameter imaging strategies:

  • Co-stain for c-myc with its transcriptional targets

  • Perform sequential immunofluorescence for c-myc and multiple functional markers

  • Combine with FISH to correlate protein expression with gene amplification status

  • Implement spatial analysis to examine c-myc expression in different tissue microenvironments

• ChIP-based functional assays:

  • ChIP-seq to identify genomic binding sites

  • ChIP-qPCR to quantify binding to specific target genes

  • Sequential ChIP to identify co-regulatory factors

  • Integration with RNA-seq data to correlate binding with transcriptional output

• Proximity ligation assays (PLA):

  • Detect interactions between c-myc and co-factors

  • Visualize associations with transcriptional machinery

  • Quantify interactions with regulatory proteins

• Live-cell approaches:

  • Use c-myc antibody fragments for live-cell imaging

  • Combine with reporters of transcriptional activity

  • Monitor dynamic changes in response to perturbations

When interpreting results, consider that c-myc functions may vary significantly between cell types, as it is "reported to be widely expressed in many tissue types" but with context-dependent functions.

How can researchers optimize detection of low abundance c-myc in primary cells?

Detecting low abundance c-myc in primary cells presents unique challenges that require specialized approaches:

• Sample preparation optimization:

  • Process samples immediately after collection

  • Use protease and phosphatase inhibitor cocktails

  • Consider nuclear extraction to concentrate c-myc protein

  • Implement gentle lysis conditions that preserve epitope integrity

• Western blot sensitivity enhancement:

  • Increase protein loading (50-100 μg)

  • Use high-sensitivity detection reagents (enhanced ECL substrates)

  • Implement longer exposure times

  • Consider signal amplification systems (biotin-streptavidin)

  • Optimize transfer conditions for efficient protein transfer

• Immunofluorescence sensitivity improvements:

  • Tyramide signal amplification (TSA) systems increase detection sensitivity 10-100 fold

  • Extend primary antibody incubation (overnight or longer at 4°C)

  • Use higher primary antibody concentrations with extensive washing

  • Implement antigen retrieval methods even for non-FFPE samples

  • Use bright, photostable fluorophores and sensitive detection systems

• Flow cytometry optimization:

  • Thorough permeabilization for nuclear antigen access

  • Use of bright fluorophores (PE, APC) rather than FITC

  • Extended antibody incubation times

  • Higher antibody concentrations with validated specificity

  • Careful gating strategies with appropriate controls

• General considerations:

  • Always run positive controls (cell lines with known c-myc expression)

  • Include appropriate negative controls

  • Consider batch processing to minimize technical variation

  • Implement quantitative image analysis for IF/IHC applications

The search results indicate successful c-myc detection in cancer cell lines, which typically have higher expression than primary cells , underscoring the need for enhanced sensitivity when working with primary samples.

What strategies can differentiate physiological from pathological c-myc expression?

Distinguishing normal from aberrant c-myc expression is crucial for understanding its role in disease:

• Quantitative assessment approaches:

  • Digital pathology with precise nuclear intensity quantification

  • Flow cytometry with absolute protein quantification

  • Quantitative Western blotting with standard curves

  • Single-cell analysis to identify abnormal subpopulations

• Context-specific normalization:

  • Compare against appropriate tissue-matched controls

  • Account for proliferation status (c-myc normally increases during proliferation)

  • Consider developmental stage and tissue-specific expression patterns

  • Normalize to internal controls within the same sample

• Multi-parameter analysis:

  • Co-staining for c-myc with markers of cellular stress

  • Assessment of subcellular localization patterns

  • Correlation with post-translational modifications

  • Integration with genomic analysis (FISH for amplification status)

• Functional correlations:

  • Monitor c-myc target gene expression

  • Assess relationship with proliferation markers

  • Analyze response to differentiation signals

  • Examine correlation with pathway activation markers

The search results note that c-myc dysregulation is associated with "a variety of hematopoietic tumors, leukemias and lymphomas, including Burkitt lymphoma" , highlighting the importance of distinguishing physiological from pathological expression in diagnostic and research applications.

• Threshold determination:

  • Establish tissue-specific thresholds for normal expression

  • Consider heterogeneity within samples

  • Validate findings across multiple experimental approaches

  • Correlate with clinical parameters in patient samples

How should researchers address inconsistent c-myc detection in Western blotting?

Inconsistent c-myc Western blot results can stem from several factors. Here's a systematic troubleshooting approach:

• Sample preparation issues:

  • Protein degradation: Add protease inhibitors and maintain samples at 4°C

  • Incomplete extraction: Optimize lysis buffers for nuclear proteins

  • Phosphorylation variability: Include phosphatase inhibitors

  • Variable expression: Standardize culture/treatment conditions

• Technical variables:

  • Protein loading inconsistency: Verify equal loading with housekeeping proteins

  • Transfer efficiency: Confirm complete transfer with reversible staining

  • Blocking optimization: Test different blocking agents (milk vs. BSA)

  • Antibody performance: The search results show that antibody dilutions of "1/1000" are commonly recommended—verify your working concentration

• Detection challenges:

  • Band size verification: c-myc typically appears at "57-62 kDa" despite theoretical molecular weight of 49 kDa

  • Multiple bands: May represent isoforms or degradation products

  • Signal strength: Adjust exposure times or consider more sensitive detection systems

  • Background issues: Optimize washing procedures and blocking conditions

• Validation approaches:

  • Multiple antibodies: Test several c-myc antibodies targeting different epitopes

  • Positive controls: Include lysates from cells with verified c-myc expression

  • Negative controls: Consider c-myc knockout/knockdown samples as available

  • Loading controls: Use multiple housekeeping proteins for normalization

• Methodological adjustments:

  • Fresh antibody aliquots: Avoid repeated freeze-thaw cycles

  • Optimization of incubation times and temperatures

  • Protocol consistency between experiments

  • Careful recording of lot numbers and reagent sources

These approaches address the most common sources of variability in c-myc Western blotting.

What strategies can overcome background issues in c-myc immunofluorescence?

High background in c-myc immunofluorescence staining can obscure specific signals. Here are effective strategies to improve signal-to-noise ratio:

• Fixation and permeabilization optimization:

  • Test different fixatives (4% PFA vs. methanol)

  • Adjust fixation duration (10-20 minutes)

  • Optimize permeabilization agent concentration (0.1-0.5% Triton X-100)

  • Include a post-fixation washing step with glycine to quench reactive aldehydes

• Blocking improvements:

  • Extend blocking time (1-2 hours)

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Include detergents in blocking solution (0.1% Triton X-100 or Tween-20)

  • Consider dual blocking with serum and BSA

• Antibody optimization:

  • Careful antibody titration (starting with manufacturer recommendations)

  • Extended washing steps (5-6 washes of 5-10 minutes each)

  • Pre-adsorption of antibodies with cell/tissue lysates

  • Test different antibody clones and formats

• Autofluorescence reduction:

  • Sudan Black B treatment post-immunostaining

  • Commercial autofluorescence quenchers

  • Selection of fluorophores away from autofluorescence spectrum

  • Spectral unmixing during image acquisition

• Controls and validation:

  • Include secondary-only controls

  • Peptide competition assays to verify specificity

  • Isotype controls at matching concentrations

  • Positive control samples with known staining patterns

The search results show successful staining in fixed HeLa cells with specific nuclear localization , which provides a useful benchmark for optimizing your protocol.

How can researchers address c-myc antibody cross-reactivity issues?

Cross-reactivity can compromise experiment specificity. Here's how to identify and mitigate c-myc antibody cross-reactivity:

• Identification of cross-reactivity:

  • Unexpected bands on Western blots

  • Non-nuclear staining patterns in immunofluorescence

  • Signal in negative control samples (knockout/knockdown)

  • Discrepancies between results with different antibodies

• Validation strategies:

  • Test antibody in c-myc knockout/knockdown systems

  • The search results show this approach: "Western blot shows lysates of HEK293T human embryonic kidney parental cell line and c-Myc knockout HEK293T cell line (KO)"

  • Peptide competition assays to confirm specificity

  • Compare multiple antibodies targeting different epitopes

  • Pre-adsorption with potential cross-reactive proteins

• Technical optimizations:

  • Increase washing stringency (longer washes, higher detergent concentration)

  • Optimize blocking conditions to reduce non-specific binding

  • Adjust antibody concentration to improve signal-to-noise ratio

  • Consider more specific monoclonal antibodies

• Alternative approaches:

  • Use epitope-tagged c-myc in expression systems

  • Implement orthogonal detection methods (mass spectrometry)

  • Combine antibody detection with mRNA analysis

  • Consider alternative c-myc antibody clones

• Careful data interpretation:

  • Document all observed cross-reactivity

  • Be transparent about limitations in publications

  • Use multiple validation approaches for critical findings

  • Consider biological context when interpreting results

The search results indicate the importance of antibody validation, with some products reporting extensive citation records and validation data , suggesting reliable performance in various applications.

What emerging technologies are enhancing c-myc antibody applications?

Several cutting-edge technologies are expanding the capabilities of c-myc antibody-based research:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) for high-parameter c-myc analysis with dozens of other markers

  • Microfluidic platforms for protein quantification in rare cell populations

  • Single-cell Western blotting for heterogeneity assessment

  • Spatial proteomics combining c-myc detection with subcellular localization

• Advanced imaging approaches:

  • Super-resolution microscopy revealing c-myc spatial organization within the nucleus

  • Expansion microscopy for enhanced visualization of nuclear architecture

  • Multiplexed ion beam imaging (MIBI) for highly multiplexed tissue analysis

  • Light-sheet microscopy for 3D visualization of c-myc distribution

• Proximity-based interaction mapping:

  • BioID and TurboID for in vivo proximity labeling

  • APEX2-based proximity labeling

  • Split-protein complementation assays

  • Improved PLA (Proximity Ligation Assay) variants

• Nanobody and aptamer technologies:

  • Camelid single-domain antibodies (nanobodies) against c-myc

  • Intrabodies for live-cell visualization of c-myc

  • DNA/RNA aptamers as antibody alternatives

  • Genetically encoded probes for real-time imaging

• Computational approaches:

  • Machine learning for image analysis

  • Integrative multi-omics incorporating c-myc antibody data

  • Automated high-content screening

  • Digital pathology algorithms for c-myc quantification

These emerging technologies will likely facilitate more sophisticated analysis of c-myc biology in complex systems, enabling researchers to address questions about c-myc function with unprecedented resolution and specificity.

How might next-generation antibody technologies improve c-myc research?

Next-generation antibody technologies promise to address current limitations and expand research capabilities:

• Recombinant antibody development:

  • Consistent production without batch-to-batch variation

  • Engineered binding properties for improved specificity

  • Reduced background through affinity maturation

  • Custom epitope targeting for specific research applications

• Site-specific conjugation strategies:

  • Controlled fluorophore-to-antibody ratios

  • Oriented conjugation preserving antigen-binding capacity

  • Multifunctional antibodies with multiple detection modalities

  • Enzyme-antibody conjugates for signal amplification

• Miniaturized antibody formats:

  • Fab and F(ab')2 fragments for improved tissue penetration

  • Single-chain variable fragments (scFvs) for reduced background

  • Bispecific antibodies targeting c-myc and functional markers simultaneously

  • Intrabodies compatible with live-cell imaging

• Reversible binding antibodies:

  • pH-sensitive binding for elution and reuse

  • Temperature-responsive antibodies

  • Photoswitchable binding for temporal control

  • Multiplexed epitope detection through sequential staining and elution

• Integration with CRISPR technologies:

  • Antibody-guided CRISPR targeting

  • Endogenous tagging of c-myc for antibody-independent detection

  • CUT&Tag approaches combining antibody recognition with DNA sequencing

  • Spatial transcriptomics validated with antibody detection

These advances will likely enhance the specificity, reproducibility, and versatility of c-myc detection methods, enabling more sophisticated analyses of its roles in normal physiology and disease states.

What are the standardized protocols for c-myc detection across different platforms?

Standardized protocols for c-myc detection vary by platform. Here are optimized procedures based on the search results and research best practices:

Western Blot Protocol for c-myc Detection:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease/phosphatase inhibitors

  • Sonicate briefly to shear genomic DNA

  • Centrifuge at 14,000g for 15 minutes at 4°C

  • Quantify protein concentration (BCA or Bradford assay)

• SDS-PAGE and transfer:

  • Load 20-50 μg protein per lane

  • Separate proteins on 10-12% polyacrylamide gel

  • Transfer to PVDF membrane at 100V for 90 minutes (wet transfer)

• Immunodetection:

  • Block in 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with anti-c-myc antibody at 1:1000 dilution overnight at 4°C

  • Wash 3×10 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

  • Wash 3×10 minutes with TBST

  • Develop using ECL substrate and image

• Expected results:

  • c-myc band at approximately 57-62 kDa

  • Positive controls: HeLa, HT-29, Jurkat, and LNCaP cell lines

Immunofluorescence Protocol for c-myc Detection:

• Sample preparation:

  • Culture cells on coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Immunostaining:

  • Block with 5% normal serum in PBS for 1 hour

  • Incubate with primary antibody at 3 μg/mL for 3 hours at room temperature

  • Wash 3×5 minutes with PBS

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour

  • Wash 3×5 minutes with PBS

  • Counterstain with DAPI and mount

• Expected results:

  • Specific nuclear localization of c-myc

  • HeLa cells serve as positive control

Flow Cytometry Protocol for c-myc Detection:

• Sample preparation:

  • Harvest cells and wash with PBS

  • Fix with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

• Staining procedure:

  • Block with 10% normal serum for 30 minutes

  • Incubate with anti-c-myc antibody (PE-conjugated or primary unconjugated)

  • If using unconjugated primary, incubate with fluorophore-conjugated secondary

  • Wash and resuspend in flow cytometry buffer

• Controls:

  • Isotype control antibody

  • Unstained cells

  • Single-color controls for compensation

What quantitative benchmarks exist for c-myc antibody performance?

Establishing quantitative benchmarks helps evaluate antibody performance. Here are key performance metrics based on the search results and research standards:

Western Blot Performance Metrics:

Immunofluorescence Performance Metrics:

ChIP Performance Metrics:

These benchmarks provide quantitative standards for evaluating antibody performance across applications and can help researchers troubleshoot suboptimal results.