MYC (Ab-358) Antibody

What is the MYC (Ab-358) Antibody and what epitope does it recognize?

The MYC (Ab-358) Antibody is a rabbit polyclonal antibody that detects endogenous levels of total MYC protein. It was developed using a synthetic peptide sequence around amino acids 356-360 (R-R-T-H-N) derived from Human MYC . This antibody is produced by immunizing rabbits with this peptide conjugated to KLH and is subsequently purified by affinity-chromatography using the epitope-specific peptide .

What are the validated applications for MYC (Ab-358) Antibody?

The MYC (Ab-358) Antibody has been validated for multiple experimental applications:

While not explicitly validated in the search results, researchers may consider testing this antibody for other applications such as immunoprecipitation or immunofluorescence based on their specific experimental requirements .

How does MYC (Ab-358) Antibody compare to phospho-specific MYC antibodies?

MYC (Ab-358) Antibody differs fundamentally from phospho-specific MYC antibodies in terms of epitope recognition and experimental applications:

Research has shown that phosphorylation at Thr358 by PAK2 negatively regulates c-MYC activity by inhibiting DNA binding . When designing experiments, researchers should consider whether they need to measure total MYC levels or specific phosphorylated forms that indicate particular activation states.

For comprehensive studies of MYC function, using both types of antibodies in parallel can provide valuable insights into both MYC expression levels and its post-translational regulation in experimental systems.

What are the recommended storage and handling conditions for MYC (Ab-358) Antibody?

Proper storage and handling are essential for maintaining antibody activity:

• The antibody is supplied at 1.0mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

• For long-term preservation, store at -20°C .

• For short-term use, storage at 4°C is acceptable .

• Avoid repeated freeze-thaw cycles as these can degrade the antibody .

• Consider aliquoting into smaller volumes to minimize freeze-thaw cycles when designing long-term experiments.

Due to the presence of sodium azide in the storage buffer, proper disposal and handling precautions should be observed as sodium azide is toxic and can form explosive compounds in metal plumbing.

How can MYC (Ab-358) Antibody be used to study MYC's role in cancer biology?

MYC (Ab-358) Antibody is particularly valuable for cancer research applications since MYC expression is significantly elevated in almost all cancers including hepatocellular carcinoma (HCC) and glioblastoma (GBM) . Several methodological approaches can be employed:

• Comparative expression analysis: Measure MYC protein levels between normal and cancerous tissues using Western blotting or IHC to establish correlation with malignant transformation.

• Signaling pathway studies: Investigate how MYC interacts with other oncogenic pathways. Research findings demonstrate that MYC overexpression deregulates the androgen receptor (AR) transcriptional program, which is a driving force in prostate cancer .

• Growth and proliferation studies: Monitor MYC levels during cell proliferation experiments, as MYC is an important positive regulator of cell growth and proliferation .

• Apoptosis research: Examine MYC's role in programmed cell death, as it has been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or growth factors .

• Therapeutic response monitoring: Assess changes in MYC expression following experimental treatments, such as in studies examining 5-lipoxygenase inhibition which has been shown to down-regulate c-Myc expression and trigger proteasomal degradation in cancer cells .

What controls should be included when using MYC (Ab-358) Antibody?

Proper experimental controls are critical for generating reliable and interpretable results:

• Positive controls: Include cell lines with confirmed MYC expression, such as LNCaP-MYC cells or other cancer cell lines known to overexpress MYC.

• Negative controls: Utilize cells with MYC knockdown via siRNA. The ON-TARGETplus Human MYC SMARTpool siRNA mentioned in the search results provides a validated approach for creating MYC-depleted control samples .

• Loading controls: For Western blotting, include housekeeping protein controls (β-actin, GAPDH, etc.) to normalize protein amounts across samples.

• Isotype controls: For IHC applications, include normal rabbit IgG at the same concentration as the primary antibody to assess non-specific binding.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity of the observed signal.

• Cross-validation: Compare results with another validated anti-MYC antibody targeting a different epitope to provide additional confidence in result specificity.

How can MYC (Ab-358) Antibody be used to study MYC-dependent transcriptional regulation?

MYC functions primarily as a transcription factor that regulates gene expression through binding to specific DNA sequences. MYC (Ab-358) Antibody can be employed to study this function through several approaches:

• Chromatin Immunoprecipitation (ChIP): While not explicitly validated for ChIP in the search results, the antibody could potentially be used to immunoprecipitate MYC-bound chromatin, followed by sequencing or PCR of bound DNA regions.

• E-box binding studies: Research findings indicate that MYC binds to both canonical E-box 2 and non-canonical E-box 3 elements with different regulatory outcomes . The antibody could be used in conjunction with EMSA (Electrophoretic Mobility Shift Assay) and supershift analysis to confirm MYC binding to specific E-box elements.

• Expression correlation studies: Combine MYC protein detection with RNA analysis of target genes. For example, research has shown that MYC inhibits MC-let-7a-1~let-7d microRNA cluster in some cancer types through binding to non-canonical E-box 3 .

• DNA binding domain studies: Investigate how MYC's DNA binding activity is regulated. Research findings suggest that phosphorylation at Thr358 by PAK2 negatively regulates c-MYC activity by inhibiting DNA binding .

How can experimental conditions be optimized to improve signal-to-noise ratio with MYC (Ab-358) Antibody?

Optimizing signal-to-noise ratio requires attention to multiple experimental parameters:

• Sample preparation optimization:

  • For Western blotting: Use proper lysis buffers with protease inhibitors

  • For IHC: Optimize fixation times and antigen retrieval methods

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum)

  • Optimize blocking time and temperature

• Antibody dilution titration:

  • Start with recommended dilutions (1:500-1:1000 for WB, 1:50-1:100 for IHC)

  • Perform serial dilutions to identify optimal concentration

• Incubation conditions:

  • Test different temperatures (4°C, room temperature)

  • Vary incubation times (overnight vs. 1-4 hours)

• Washing optimization:

  • Increase washing buffer stringency with appropriate detergent concentrations

  • Extend washing times and number of washes

• Detection system selection:

  • Choose appropriate secondary antibodies (several options are provided for Anti-Rabbit IgG H&L in various conjugates)

  • Select detection chemistry based on expected signal strength

Can MYC (Ab-358) Antibody be used to study post-translational modifications of MYC?

• Comparative analysis: Run parallel Western blots with MYC (Ab-358) Antibody and modification-specific antibodies like Anti-c-Myc (phospho Thr358) to compare total versus modified MYC levels .

• Sequential immunoprecipitation: Immunoprecipitate with MYC (Ab-358) Antibody followed by Western blotting with modification-specific antibodies to determine the proportion of modified MYC in the total pool.

• Ubiquitination studies: MYC undergoes ubiquitination by the SCF(FBXW7) complex when phosphorylated at Thr-58 and Ser-62, leading to its degradation by the proteasome . Researchers can use MYC (Ab-358) Antibody to monitor total MYC levels in experiments with proteasome inhibitors.

• Phosphorylation analysis: Use phosphatase treatment of samples followed by Western blotting with MYC (Ab-358) Antibody to assess the impact of phosphorylation on MYC stability or mobility.

What research models have been validated with MYC (Ab-358) Antibody?

Based on the search results, MYC (Ab-358) Antibody has been validated in several research models:

• Human cell lines: The antibody shows reactivity with human samples (labeled as "Hu" in specifications) .

• Cancer cell models: Several relevant cancer models appear in the search results where MYC function has been studied:

  • Prostate cancer: LNCaP-MYC and corresponding empty vector (EV) lines

  • Hepatocellular carcinoma: HepG2 cells

  • Normal liver cells: L02

  • Glioblastoma: U87 and U251 cells

• Primary tissue samples: While not explicitly mentioned, the antibody is likely suitable for human primary tissue samples based on its validated IHC application.

For researchers planning to use this antibody in novel models, preliminary validation experiments should be conducted, including positive and negative controls, siRNA knockdown of MYC to confirm specificity, and comparison with other validated MYC antibodies.

How can MYC (Ab-358) Antibody be used to study cell cycle regulation by MYC?

MYC (Ab-358) Antibody can be employed to examine MYC's role in cell cycle regulation through several methodological approaches:

• Cell cycle phase analysis: Monitor MYC protein levels across different cell cycle phases using synchronized cell populations. Research indicates that MYC expression is generally induced after mitogenic stimulation or serum induction .

• Proliferation studies: Combine MYC detection with proliferation markers. Search results mention using BrdU incorporation to assess DNA synthesis in relation to MYC activity .

• Growth factor dependency: As MYC can induce apoptosis when expressed in the absence of serum or growth factors , the antibody can be used to correlate MYC levels with cell survival under various growth factor conditions.

• Metabolic regulation: MYC is implicated in glucose metabolism regulation. Research findings suggest that glucose can drive growth factor-independent cancer proliferation, potentially through MYC-mediated mechanisms .

• Genetic manipulation studies: Implement MYC knockdown or overexpression followed by cell cycle analysis, while confirming the manipulation's effectiveness with MYC (Ab-358) Antibody.

How does the MYC (Ab-358) Antibody perform in protein array and high-throughput screening applications?

The search results reference use of MYC (Ab-358) Antibody in protein array applications, suggesting compatibility with high-throughput screening formats:

• Antibody microarrays: Search result mentions "Phospho Explorer Antibody Microarray" which includes MYC (Ab-358) with a fold change value of 0.93, indicating this antibody can be incorporated into microarray formats.

• Multi-protein detection: The antibody can likely be used in multiplex detection systems where multiple proteins are analyzed simultaneously, though specific validation for such applications would be advisable.

• Screening applications: When designing high-throughput screens, researchers should consider appropriate controls and standardization methods to ensure consistent performance across multiple samples.

• Quantification considerations: For accurate quantification in array formats, appropriate normalization controls and statistical analysis methods should be implemented to account for technical variability.

What methodological considerations are important when using MYC (Ab-358) Antibody in immunohistochemistry?

When employing MYC (Ab-358) Antibody for immunohistochemistry, several methodological considerations are critical:

• Fixation protocol: Optimized fixation is essential - typically 10% neutral buffered formalin for 24-48 hours for tissue samples.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions.

• Dilution optimization: Begin with the recommended dilution range of 1:50-1:100 and adjust based on signal intensity and background.

• Detection system selection: Consider amplification systems for low-abundance targets, but be cautious of increased background.

• Counterstaining: Choose appropriate counterstains that do not interfere with the primary antibody signal.

• Tissue-specific considerations: Different tissue types may require specific modifications to the protocol due to varying protein abundance and tissue composition.

• Controls: Include positive control tissues known to express MYC, negative control tissues, and antibody control slides (omitting primary antibody) in each staining run.

Can MYC (Ab-358) Antibody distinguish between different MYC family members?

The MYC family contains several related proteins including c-MYC, N-MYC, and L-MYC, which share structural homology but have distinct expression patterns and functions:

• Specificity analysis: The antibody detects endogenous levels of total MYC protein , but the search results do not explicitly state cross-reactivity testing against other MYC family members.

• Epitope conservation: The epitope region (aa 356-360, R-R-T-H-N) should be compared across MYC family members to predict potential cross-reactivity.

• Experimental verification: Researchers studying multiple MYC family members should perform validation experiments using cell lines with differential expression of specific MYC family members.

• Alternative approaches: For applications requiring absolute specificity between MYC family members, complementary techniques such as RNA analysis (RT-PCR, RNA-seq) may provide additional confirmation of which family member is being expressed.

• Positive controls: When testing for specificity, include positive controls with confirmed expression of different MYC family members.

What is the relationship between MYC detection by this antibody and MYC-targeted cancer therapies?

Understanding MYC protein levels and activity is crucial for developing and evaluating MYC-targeted cancer therapies:

• Therapeutic monitoring: MYC (Ab-358) Antibody can be used to monitor MYC protein levels in response to experimental therapeutics. For example, research has shown that inhibition of 5-lipoxygenase selectively triggers disruption of c-Myc signaling in cancer cells but not in normal fibroblasts .

• Biomarker development: The antibody can help establish MYC as a biomarker for predicting therapeutic response or disease progression in clinical samples.

• Mechanism of action studies: Researchers can use the antibody to investigate how novel compounds affect MYC stability, localization, or activity. For instance, research findings indicate that MYC constitutes the germinal center B cell division timer that when deregulated leads to emergence of B cell lymphoma .

• Resistance mechanisms: By monitoring MYC expression in treatment-resistant cells, researchers can elucidate potential resistance mechanisms involving MYC deregulation.

• Combination therapy development: The antibody can help identify effective combination therapies by revealing how different drugs impact MYC expression and activity in concert.