Myc (PMYCSHG) Antibody

What is the Myc (PMYCSHG) antibody and what are its primary research applications?

Myc (PMYCSHG) antibody is a mouse monoclonal antibody produced by immunizing mice with a synthetic peptide (EQKLISEEDL) coupled to KLH. This antibody specifically recognizes the c-Myc protein, which functions as a transcription factor regulating cell cycle progression, apoptosis, and cellular transformation . The antibody is primarily used in Western Blotting (recommended concentration of 0.5μg/ml) and Immunoprecipitation applications . The antibody belongs to the Mouse IgG1 subclass and is formulated in 1×PBS with 50% glycerol for stability .

How does c-Myc function as a transcription factor in cellular processes?

c-Myc functions as a transcription factor that binds DNA both non-specifically and specifically recognizes the core sequence 5'-CAC[GA]TG-3' . It activates the transcription of growth-related genes and binds to the VEGFA promoter, thereby promoting VEGFA production and subsequent sprouting angiogenesis . Additionally, c-Myc regulates somatic reprogramming and controls self-renewal of embryonic stem cells . The protein works with TAF6L to activate target gene expression through RNA polymerase II pause release . c-Myc also positively regulates transcription of HNRNPA1, HNRNPA2, and PTBP1, which in turn regulate splicing of pyruvate kinase PKM, contributing to the production of the PKM M2 isoform that is characteristic of many cancer cells .

What is the molecular weight range for c-Myc protein detection and what band patterns should researchers expect in Western blotting?

When using c-Myc antibodies for Western blotting, researchers should expect to detect bands in the molecular weight range of 57-70 kDa . The specific band pattern may vary depending on the cell type being analyzed and post-translational modifications of the c-Myc protein. For optimal results when conducting Western blot experiments, a recommended antibody dilution of 1:1000 is suggested . When conducting Simple Western™ assays, a dilution range of 1:10 to 1:50 is typically recommended . Band intensity can vary based on expression levels of c-Myc, which are known to fluctuate significantly across different cell types and under different cellular conditions.

How should researchers optimize Western blotting protocols for c-Myc detection using the PMYCSHG antibody?

For optimal Western blotting using the Myc (PMYCSHG) antibody, researchers should follow these methodological steps:

• Sample preparation: Extract proteins under conditions that preserve phosphorylation states if studying phosphorylated forms of c-Myc.

• Loading control: Include appropriate loading controls as c-Myc expression can vary significantly between samples.

• Antibody concentration: Start with the recommended 0.5μg/ml concentration for Western blotting .

• Blocking: Use a 5% BSA in TBST solution to minimize background.

• Incubation time: Incubate with primary antibody overnight at 4°C for optimal binding.

• Detection system: Use an HRP-conjugated secondary antibody specific to mouse IgG1.

• Visualization: For low abundance samples, consider enhanced chemiluminescence (ECL) detection systems.

The PMYCSHG antibody has been validated to detect c-Myc in human cervical epithelial carcinoma (HeLa), colon adenocarcinoma (HT-29), acute T cell leukemia (Jurkat), and prostate cancer (LNCaP) cell lines , making it versatile for various cancer research applications.

What are the recommended protocols for using Myc (PMYCSHG) antibody in immunoprecipitation experiments?

When conducting immunoprecipitation (IP) with the Myc (PMYCSHG) antibody, researchers should implement the following protocol for optimal results:

• Cell lysis: Use a non-denaturing lysis buffer (e.g., 1% NP-40, 150mM NaCl, 50mM Tris pH 8.0) supplemented with protease and phosphatase inhibitors.

• Pre-clearing: Pre-clear cell lysates with protein G beads to reduce non-specific binding.

• Antibody amount: Use the antibody at a 1:50 dilution for immunoprecipitation .

• Incubation: Incubate the antibody with cell lysate overnight at 4°C with gentle rotation.

• Bead binding: Add protein G beads and incubate for 2-4 hours at 4°C.

• Washing: Perform at least four washes with lysis buffer to remove non-specific interactions.

• Elution: Elute bound proteins using SDS sample buffer heated to 95°C for 5 minutes.

• Analysis: Analyze immunoprecipitated samples by Western blotting using a different c-Myc antibody to confirm specificity.

For co-immunoprecipitation experiments investigating c-Myc binding partners, maintain physiological salt concentrations and consider using crosslinking agents to stabilize transient interactions.

What is the optimal approach for validating c-Myc (PMYCSHG) antibody specificity in experimental systems?

To validate the specificity of the Myc (PMYCSHG) antibody, researchers should employ multiple complementary approaches:

• Knockout/knockdown controls: Use c-Myc knockout or knockdown cell lines to confirm the absence of signal when the target protein is not present .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (EQKLISEEDL) before application to samples, which should abolish specific binding.

• Multiple antibody comparison: Use alternative c-Myc antibodies that recognize different epitopes and compare detection patterns.

• Recombinant protein controls: Include purified c-Myc recombinant protein as a positive control.

• Cross-reactivity testing: Test the antibody against related proteins (e.g., N-Myc, L-Myc) to ensure specificity.

• Mass spectrometry validation: Identify proteins in immunoprecipitated samples using mass spectrometry to confirm c-Myc presence.

• Size verification: Confirm that detected bands match the expected molecular weight (57-70 kDa) .

Some c-Myc antibodies have been knockout (KO) validated , which provides the highest level of specificity confirmation and should be preferred when available.

How can the Myc (PMYCSHG) antibody be used to study bimodal expression patterns in T cell activation?

The PMYCSHG antibody can be effectively employed to investigate the bimodal expression patterns of c-Myc during T cell activation using these methodological approaches:

• Flow cytometry analysis: Use the antibody in combination with T cell activation markers like CD69 to observe the bimodal (on/off) pattern of Myc expression that is maintained during sustained T cell responses to p/MHC ligands .

• Time-course experiments: Track Myc expression at different time points (early activation, sustained activation) to observe that within the first few hours of activation, there is an increase in the percentage of T cells expressing Myc but no increase in the maximal amount of Myc expressed per cell .

• Dose-response studies: Compare TCR ligand dose-response for induction of Myc versus CD69, noting that upregulation of CD69 is more sensitive to TCR ligand dose than Myc induction .

• Population analysis: Observe that Myc expression is restricted to CD69-expressing T cells and shows a bimodal distribution in CD69-positive TCR-activated lymphocytes .

• Single-cell analysis: Use imaging techniques with the antibody to examine heterogeneity in Myc expression levels within responding T cell populations.

This approach can provide insights into the threshold-dependent regulation of Myc expression during immune responses and its relationship to T cell activation and proliferation decisions.

What are the technical considerations for chromatin immunoprecipitation (ChIP) experiments using c-Myc antibodies?

For successful chromatin immunoprecipitation (ChIP) experiments investigating c-Myc binding to target genes, researchers should consider the following technical guidelines:

• Chromatin preparation: Use 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation for optimal results .

• Antibody amount: Use 10 μl of antibody per ChIP reaction .

• Crosslinking conditions: Optimize formaldehyde crosslinking time (typically 10-15 minutes) to preserve protein-DNA interactions without over-crosslinking.

• Sonication parameters: Carefully optimize sonication conditions to generate DNA fragments of 200-500 bp.

• Controls:

  • Include a negative control using non-specific IgG

  • Use a positive control antibody against a known abundant transcription factor

  • Include primer sets for known c-Myc target genes and non-target regions

• Validation: Validate ChIP results using multiple c-Myc antibodies targeting different epitopes.

• Data analysis: Normalize to input and IgG controls when analyzing qPCR data from ChIP experiments.

The c-Myc antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits , which provides a standardized protocol that researchers can follow to enhance reproducibility.

How can researchers investigate the relationship between c-Myc phosphorylation at S62 and its functional activity?

To study the relationship between c-Myc phosphorylation at S62 and its functional activity, researchers can implement the following experimental approaches:

• Phospho-specific antibody detection: Use phospho-S62 specific c-Myc antibodies (like ab51156) in Western blotting to determine phosphorylation status under different cellular conditions.

• Mutagenesis studies: Generate S62A (phospho-deficient) and S62D/E (phospho-mimetic) c-Myc mutants and compare their functional activities in:

  • Transcriptional reporter assays

  • Cellular proliferation and apoptosis assays

  • DNA binding capacity using electrophoretic mobility shift assays (EMSA)

• Kinase inhibition experiments: Use inhibitors of kinases known to phosphorylate S62 (e.g., ERK, CDKs) to evaluate their impact on c-Myc activity and stability.

• Phosphatase studies: Investigate the role of PP2A, which dephosphorylates S62, using phosphatase inhibitors or siRNA-mediated knockdown.

• Cell cycle synchronization: Analyze S62 phosphorylation levels throughout the cell cycle using synchronized cell populations.

• Dual phosphorylation analysis: Investigate the interplay between S62 phosphorylation and T58 phosphorylation, as these modifications have opposing effects on c-Myc stability.

This approach enables researchers to elucidate how S62 phosphorylation affects c-Myc stability, protein-protein interactions, and transcriptional activity in normal and disease states.

What are common issues in Western blotting with c-Myc antibodies and how can they be resolved?

Researchers frequently encounter several challenges when conducting Western blotting with c-Myc antibodies. Here are common issues and their solutions:

When working with c-Myc antibodies, it's essential to use fresh samples and optimize each step of the Western blotting protocol for your specific experimental system.

How can researchers differentiate between specific and non-specific binding when using Myc (PMYCSHG) antibody?

To accurately differentiate between specific and non-specific binding when using the Myc (PMYCSHG) antibody, researchers should implement these validation strategies:

• Positive and negative controls:

  • Use cell lines known to express high levels of c-Myc (positive control, e.g., Jurkat, HeLa)

  • Include c-Myc-negative or knockdown samples (negative control)

• Competing peptide assay:

  • Pre-incubate the antibody with excess immunizing peptide (EQKLISEEDL)

  • Specific binding should be abolished while non-specific binding persists

• Multiple antibody approach:

  • Compare staining patterns using different c-Myc antibodies targeting distinct epitopes

  • Consistent patterns across antibodies indicate specific binding

• Band verification:

  • Confirm that observed bands match the expected molecular weight (57-70 kDa)

  • Use protein ladders and positive control samples for accurate size determination

• Signal quantification:

  • Plot signal-to-noise ratios under different antibody concentrations

  • Specific binding shows dose-dependent saturation while non-specific binding increases linearly

• Secondary antibody controls:

  • Include samples with secondary antibody only to identify background signals

  • Use isotype-matched control antibodies to identify Fc receptor-mediated binding

These approaches provide complementary evidence to distinguish specific c-Myc detection from artifacts and background signals.

What are the key considerations for preserving c-Myc protein integrity during sample preparation?

c-Myc protein is notoriously labile and prone to rapid degradation, making proper sample handling critical for accurate experimental results. Here are essential considerations for preserving c-Myc integrity:

• Cell harvesting and lysis:

  • Harvest cells rapidly to prevent stress-induced changes in c-Myc levels

  • Keep samples cold (4°C) throughout processing

  • Use lysis buffers containing robust protease inhibitor cocktails

  • Include phosphatase inhibitors to preserve phosphorylation status, especially for studies involving pS62-Myc

• Buffer composition:

  • Use denaturing buffers containing SDS for complete protein extraction

  • Include reducing agents (e.g., DTT, β-mercaptoethanol) to maintain protein structure

  • Optimize detergent concentration to effectively solubilize nuclear proteins

• Storage considerations:

  • Avoid repeated freeze-thaw cycles of protein samples

  • Store samples at -80°C with protease inhibitors

  • Consider snap-freezing cell pellets in liquid nitrogen prior to lysis

• Timing factors:

  • Process samples immediately after collection

  • Minimize the time between cell lysis and denaturation in sample buffer

  • When necessary, use proteasome inhibitors (e.g., MG132) to prevent c-Myc degradation

• Handling nuclear proteins:

  • Use specialized nuclear extraction protocols for optimal c-Myc recovery

  • Consider sequential extraction methods that efficiently isolate nuclear transcription factors

Adhering to these sample preparation guidelines significantly improves the consistency and reliability of c-Myc detection in research applications.

How can researchers utilize the Myc (PMYCSHG) antibody in multiplex immunofluorescence studies of tumor microenvironments?

For multiplex immunofluorescence studies examining c-Myc expression in tumor microenvironments, researchers should implement the following methodological approach:

• Panel design:

  • Combine c-Myc (PMYCSHG) antibody with markers for different cell types (e.g., CD45 for immune cells, CD31 for endothelial cells)

  • Include functional markers (Ki67 for proliferation, cleaved caspase-3 for apoptosis)

  • Add tumor-specific markers relevant to your cancer model

• Technical optimization:

  • Determine optimal antibody concentration and incubation time for immunofluorescence applications

  • Test various antigen retrieval methods to maximize signal while preserving tissue architecture

  • Establish a sequential staining protocol to avoid cross-reactivity between antibodies

• Multiplexing strategies:

  • Use spectrally distinct fluorophores for each antibody

  • Consider tyramide signal amplification (TSA) for detecting low-abundance proteins

  • Implement sequential staining with antibody stripping between rounds for highly multiplexed panels

• Analysis approaches:

  • Employ computational image analysis to quantify c-Myc expression levels in different cell populations

  • Use machine learning algorithms to identify spatial relationships between c-Myc-expressing cells and other cell types

  • Correlate c-Myc expression patterns with clinical outcomes and treatment responses

This approach enables researchers to characterize c-Myc expression within the complex cellular context of tumors, providing insights into its role in cancer progression and potential therapeutic targeting.

What are the emerging applications of c-Myc antibodies in studying immunometabolism and T cell function?

Emerging applications of c-Myc antibodies in immunometabolism and T cell function research include:

• Metabolic reprogramming analysis:

  • Use c-Myc antibodies to correlate c-Myc expression with metabolic enzyme levels (e.g., glycolytic enzymes, glutaminase)

  • Study how c-Myc regulates the switch from oxidative phosphorylation to aerobic glycolysis during T cell activation

  • Investigate the relationship between c-Myc expression and nutrient transporter upregulation following T cell receptor stimulation

• Single-cell analyses:

  • Combine flow cytometry with c-Myc antibodies to examine the bimodal distribution of c-Myc expression in T cell populations

  • Correlate c-Myc levels with functional markers and metabolic parameters at the single-cell level

  • Use c-Myc as a marker for identifying metabolically active T cell subsets

• T cell exhaustion studies:

  • Examine how c-Myc expression patterns change during T cell exhaustion in chronic infections and cancer

  • Correlate loss of c-Myc expression with exhaustion marker upregulation (PD-1, TIM-3, LAG-3)

  • Investigate how immunotherapies affect c-Myc expression in tumor-infiltrating lymphocytes

• mTOR signaling integration:

  • Study the interplay between c-Myc and mTOR signaling in controlling T cell metabolic programming

  • Investigate how nutrient sensing pathways regulate c-Myc protein stability and function

  • Examine how metabolic interventions affect c-Myc-dependent T cell functions

These applications provide new insights into how c-Myc coordinates metabolic reprogramming with effector functions in T cells, potentially informing novel immunotherapeutic approaches.

How can researchers leverage phospho-specific c-Myc antibodies to study the dynamics of post-translational modifications in cancer progression?

Researchers can leverage phospho-specific c-Myc antibodies, particularly those targeting phospho-S62 sites , to investigate the complex dynamics of post-translational modifications in cancer progression through these methodological approaches:

• Temporal analysis of phosphorylation patterns:

  • Track changes in c-Myc phosphorylation at S62 during cell cycle progression and cancer development

  • Correlate phosphorylation patterns with c-Myc protein stability and transcriptional activity

  • Use phospho-specific antibodies in time-course experiments following growth factor stimulation

• Signaling pathway integration:

  • Map kinase cascades that regulate c-Myc phosphorylation in different cancer types

  • Study how oncogenic mutations affect c-Myc phosphorylation status

  • Investigate cross-talk between phosphorylation at S62 and other modifications (T58 phosphorylation, K48/K63 ubiquitination)

• Phosphorylation-dependent interactome analysis:

  • Use phospho-specific c-Myc antibodies for immunoprecipitation followed by mass spectrometry

  • Identify proteins that preferentially interact with phosphorylated versus non-phosphorylated c-Myc

  • Characterize how phosphorylation affects chromatin binding and transcriptional complex formation

• Therapeutic response prediction:

  • Develop phospho-c-Myc signatures that predict response to targeted therapies

  • Monitor changes in c-Myc phosphorylation patterns following treatment with kinase inhibitors

  • Correlate phosphorylation status with clinical outcomes and resistance mechanisms

This comprehensive approach enables researchers to understand how post-translational modifications of c-Myc contribute to its oncogenic functions and potentially identify new therapeutic vulnerabilities in cancer.