Mono-Methyl-TP53(Lys370) Monoclonal Antibody

What is the significance of p53 Lys370 monomethylation in cancer research?

Monomethylation of p53 at Lysine 370 represents a critical post-translational modification that influences p53 tumor suppressor function. SMYD2 (SET and MYND domain-containing protein 2) preferentially monomethylates p53 at Lys-370, which has been shown to regulate p53 activity . This methylation event creates a molecular basis for p53 tumor suppression modulation, making it a significant target in cancer research. The level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo, suggesting its role in p53 regulation pathways .

What are the primary applications for Mono-Methyl-TP53(Lys370) antibodies?

Mono-Methyl-TP53(Lys370) antibodies are primarily used in the following applications:

These applications allow researchers to study the presence, abundance, and localization of monomethylated p53 in various experimental contexts .

How do monoclonal and polyclonal Mono-Methyl-TP53(Lys370) antibodies differ in research applications?

Both monoclonal and polyclonal antibodies targeting Mono-Methyl-TP53(Lys370) are available, each with distinct characteristics:

Monoclonal antibodies offer higher specificity and reproducibility for applications requiring precise epitope recognition, while polyclonal antibodies may provide stronger signals due to multiple epitope recognition .

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal activity of Mono-Methyl-TP53(Lys370) antibodies:

• Store at -20°C for up to 12 months from the date of receipt

• Aliquot to avoid repeated freeze/thaw cycles that can degrade antibody performance

• Store in buffer containing 50% glycerol as a cryoprotectant

• Some formulations include stabilizers such as 0.02% sodium azide as preservative

• Buffer systems typically utilize PBS at pH 7.4 with protein stabilizers like BSA (0.5-1%)

Proper storage is critical as the antibody's ability to specifically recognize the monomethylated lysine can be compromised by improper handling or storage conditions .

How can researchers optimize Western blot protocols for detecting Mono-Methyl-TP53(Lys370)?

For optimal Western blot results with Mono-Methyl-TP53(Lys370) antibodies:

• Sample preparation:

  • Use fresh samples or those properly stored at -80°C

  • Include phosphatase and protease inhibitors in lysis buffers to prevent post-translational modification loss

• Dilution optimization:

  • Start with 1:1000 dilution for monoclonal antibodies

  • For polyclonal antibodies, begin with 1:500 dilution and adjust as needed

• Blocking optimization:

  • 5% non-fat dry milk or BSA in TBST is typically effective

  • For phospho-specific or methyl-specific antibodies, BSA is often preferred over milk

• Detection considerations:

  • Secondary antibody selection should match the host species (anti-mouse for monoclonal, anti-rabbit for polyclonal)

  • Extended exposure times may be necessary for low abundance targets

• Controls:

  • Include positive controls from cells with known p53 Lys370 methylation status

  • Consider using SMYD2 overexpression systems to increase methylation levels

What are the common challenges in detecting monomethylated p53 and how can they be addressed?

Detection of monomethylated p53 presents several challenges:

Addressing these challenges requires careful optimization of experimental conditions and appropriate controls to ensure specific detection of the monomethylated form of p53 .

How does Mono-Methyl-TP53(Lys370) antibody contribute to understanding SMYD2-p53 interactions?

Mono-Methyl-TP53(Lys370) antibodies are instrumental in elucidating the molecular mechanisms of SMYD2-p53 interactions:

• Structural insights:

  • High-resolution crystal structure studies show that SMYD2 specifically recognizes and monomethylates p53 at Lys370

  • The antibody allows detection of this specific modification in structural biology research

• Conformational changes:

  • Methylation at Lys370 induces conformational changes in both p53 and SMYD2

  • Molecular dynamics simulations reveal that Lys370 methylation enhances flexibility at nearby sites and establishes new long-range interactions between N-terminal and C-terminal domains of SMYD2

• Functional analysis:

  • The antibody enables researchers to correlate methylation status with functional outcomes

  • Studies show that mutation of EDEE residues in SMYD2 impairs both binding and enzymatic activity toward p53 Lys370

• Regulatory mechanisms:

  • The antibody helps identify conditions that regulate methylation levels

  • Research indicates that monomethylation of Lys370 affects anticorrelation movements between domains in SMYD2, which may influence its catalytic activity

What insights can be gained from comparative analysis of different p53 post-translational modifications?

Comparative analysis of different p53 post-translational modifications using specific antibodies reveals:

• Methylation crosstalk:

  • Methylation at Lys370 appears to interact differently with the SMYD2 structure compared to methylation at Lys372

  • While Lys370 methylation establishes new long-range interactions, Lys372 methylation fails to activate similar interactions

• Modification patterns in cancer:

  • Proteomic profiling reveals that TP53 mutation status correlates with distinct patterns of post-translational modifications

  • In multiple myeloma samples with TP53 mutations, specific protein expression patterns are observed that differ from wild-type TP53

• Functional outcomes:

  • Different modifications (methylation vs. acetylation) at distinct lysine residues produce varying effects on p53 function

  • While some modifications enhance transcriptional activity, others may impair p53's ability to induce apoptosis

• Structural implications:

  • Each modification may induce unique conformational changes in p53

  • These changes affect protein-protein interactions and downstream signaling pathways

How can Mono-Methyl-TP53(Lys370) antibodies be used in studying cancer therapeutic resistance?

Mono-Methyl-TP53(Lys370) antibodies provide valuable tools for studying therapeutic resistance:

• Biomarker identification:

  • Methylation status of p53 at Lys370 may serve as a biomarker for response to certain therapies

  • Changes in methylation patterns before and after treatment can be monitored using these antibodies

• Resistance mechanisms:

  • Altered methylation of p53 may contribute to therapy resistance

  • The antibody enables tracking of methylation changes during treatment and disease progression

• Combination therapy approaches:

  • Understanding p53 methylation status helps in designing rational combination therapies

  • Targeting enzymes that regulate p53 methylation (like SMYD2) may enhance efficacy of existing therapies

• Predictive modeling:

  • The structural impact of Lys370 methylation on p53-protein interactions can inform computational models predicting drug responses

  • Molecular dynamics simulations incorporating methylation data can predict structural changes affecting drug binding

How should researchers interpret variations in mono-methyl-TP53(Lys370) levels across different cell lines?

When interpreting variations in mono-methyl-TP53(Lys370) levels across different cell lines:

• Consider SMYD2 expression levels:

  • Cell lines with higher SMYD2 expression typically show increased p53 Lys370 methylation

  • Verify SMYD2 expression via Western blot or qPCR to correlate with methylation data

• Assess p53 mutation status:

  • TP53 mutational status can significantly affect methylation patterns

  • Wild-type vs. mutant p53 may show different baseline methylation and different responses to cellular stress

• Evaluate cell type-specific factors:

  • Tissue-specific patterns of p53 isoform expression may influence methylation outcomes

  • p53 isoforms are expressed in tissue-dependent manner, affecting modification patterns

• Consider cellular localization:

  • p53 methylation may vary based on subcellular localization

  • Use immunofluorescence with the antibody to determine if methylated p53 localizes differently between cell lines

• Assess other post-translational modifications:

  • Crosstalk between different modifications may explain variations

  • Compare methylation patterns with phosphorylation or acetylation status

What factors should be considered when comparing results from different detection methods using Mono-Methyl-TP53(Lys370) antibodies?

When comparing results from different detection methods:

Additional considerations include antibody affinity differences between applications, sample preparation variations, and the need for application-specific controls .

What experimental controls are essential when using Mono-Methyl-TP53(Lys370) antibodies?

Essential controls for experiments using Mono-Methyl-TP53(Lys370) antibodies include:

• Positive controls:

  • Cell lines with SMYD2 overexpression to increase Lys370 methylation

  • Synthetic methylated peptides corresponding to the p53 region around Lys370

• Negative controls:

  • SMYD2 knockout or knockdown cells

  • Unmethylated p53 peptides for antibody validation

  • Isotype control antibodies matched to the host species (mouse IgG for monoclonal, rabbit IgG for polyclonal)

• Specificity controls:

  • Peptide competition assays with methylated vs. unmethylated peptides

  • Comparison with antibodies targeting different p53 modifications

• Technical controls:

  • Secondary antibody only controls

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

  • Tissue controls with known expression patterns for IHC

• Validation approaches:

  • Correlation with SMYD2 expression levels

  • Mass spectrometry validation of methylation site specificity

  • Cross-validation using multiple antibody clones if available

How can researchers design experiments to investigate the dynamic regulation of p53 Lys370 methylation?

To investigate dynamic regulation of p53 Lys370 methylation:

• Time-course experiments:

  • Monitor methylation changes following cellular stress (DNA damage, hypoxia)

  • Sample at multiple time points (0, 1, 3, 6, 12, 24 hours) to capture dynamics

  • Compare with other p53 modifications to establish temporal relationships

• Enzyme modulation:

  • Overexpress or knock down SMYD2 to alter methylation levels

  • Investigate potential demethylases affecting Lys370

  • Use methyltransferase inhibitors to block new methylation events

• Stimulus-response studies:

  • Expose cells to various stressors (radiation, chemotherapy drugs, oxidative stress)

  • Correlate methylation changes with p53 target gene expression

  • Assess changes in protein-protein interactions following stimulation

• Structural dynamics:

  • Combine with conformation-specific antibodies to assess structural changes

  • Use molecular dynamics simulations to predict conformational changes associated with methylation

  • Investigate domain interactions influenced by methylation status

• Cell cycle analysis:

  • Synchronize cells and assess methylation status across cell cycle phases

  • Correlate with p53 activation status and cellular outcomes

  • Compare normal vs. cancer cells for differential regulation

What methodological approaches can resolve contradictory results when studying p53 Lys370 methylation?

When facing contradictory results in p53 Lys370 methylation studies:

• Antibody validation:

  • Re-validate antibody specificity using peptide competition assays

  • Compare results with multiple antibody clones targeting the same modification

  • Consider using mass spectrometry to confirm methylation status

• Contextual factors:

  • Examine cell type-specific differences that might explain contradictions

  • Consider genetic background variations (p53 status, SMYD2 expression)

  • Assess influence of culture conditions or experimental treatments

• Technical considerations:

  • Standardize sample preparation protocols across experiments

  • Optimize antibody dilutions and incubation conditions

  • Ensure consistent detection methods and quantification approaches

• Biological complexity:

  • Investigate crosstalk with other modifications (phosphorylation, acetylation)

  • Consider spatial and temporal dynamics that might explain differences

  • Examine effects of protein-protein interactions on epitope accessibility

• Integrated approaches:

  • Combine multiple methodologies (WB, IP-MS, IF, IHC)

  • Use complementary techniques to validate findings

  • Develop computational models to reconcile apparently contradictory data