Phospho-TP53 (S9) Recombinant Monoclonal Antibody

What is the Phospho-TP53 (S9) Recombinant Monoclonal Antibody and how is it produced?

The Phospho-TP53 (S9) recombinant monoclonal antibody is a highly specific immunological tool designed to recognize the human TP53 protein when phosphorylated at serine residue 9. The antibody production process follows a sophisticated multi-step protocol. Initially, genes encoding the TP53 antibody are retrieved from rabbits immunized with a synthetic peptide derived from human TP53 protein phosphorylated at S9 . These genes are then integrated into expression vectors which are subsequently introduced into mammalian suspension cells . These genetically modified cells are cultivated under carefully controlled conditions to optimize antibody production and secretion .

Following the growth phase, the antibody is isolated from the cell culture supernatant through an intricate purification process employing affinity chromatography . The final step involves rigorous quality control, where the antibody's functionality is verified through ELISA and immunofluorescence (IF) tests to confirm its specificity and binding capacity to the target phosphorylated protein .

What is the biological significance of p53 phosphorylation at S9?

Phosphorylation of p53 at S9 represents a crucial regulatory mechanism in maintaining genomic integrity and preventing cancer development . This post-translational modification plays a pivotal role in coordinating multiple cellular processes including DNA repair mechanisms, cell cycle control checkpoints, and cell fate decisions in response to various forms of stress and cellular damage .

The precise regulation of p53 through phosphorylation events is essential for normal cellular function. When this regulation becomes dysregulated, it can lead to uncontrolled cell proliferation - a hallmark observation in many cancer cells . Understanding the dynamics and consequences of S9 phosphorylation provides critical insights into both normal p53 function and its altered states in pathological conditions.

How does p53 phosphorylation compare across different residues?

While the search results focus primarily on S9, Ser269, and Thr55 phosphorylation sites, they illustrate the site-specific effects of different phosphorylation events on p53 function. Phosphorylation at Ser269, located in the S10 β-strand of p53 within the DNA-binding domain, has been found to inactivate the transcription activation function and clonogenic suppressor activity of p53 .

Phosphorylation at Thr55 functions as a molecular switch that modulates intramolecular interactions between the disordered transactivation domain and the structured DNA-binding domain . This modification significantly impacts DNA binding capabilities and controls both activation and termination phases of p53-mediated transcriptional programs during different stages of the cellular DNA damage response .

These site-specific phosphorylation events demonstrate the complex regulatory network governing p53 activity, with each modification potentially resulting in distinct functional outcomes regarding p53's ability to control gene expression, DNA binding, protein-protein interactions, and ultimately cellular fate decisions.

What are the recommended applications and dilutions for Phospho-TP53 (S9) Antibody?

The Phospho-TP53 (S9) recombinant monoclonal antibody has been validated specifically for immunofluorescence (IF) applications with a recommended dilution range of 1:20-1:200 . This information is critical for researchers to ensure optimal signal-to-noise ratio in their experiments. The table below summarizes the application information:

Researchers should conduct preliminary titration experiments to determine the optimal antibody concentration for their specific experimental conditions, cell types, and detection systems. It is advisable to include appropriate positive and negative controls to validate staining patterns and confirm specificity.

What experimental approaches can be used to study p53 phosphorylation?

Multiple sophisticated experimental techniques have been developed to investigate p53 phosphorylation states and their functional consequences. Based on the methodologies described in the literature, researchers can employ:

• Phospho-specific antibodies - Like the Phospho-TP53 (S9) antibody, these recognize specific phosphorylated residues and can be used in techniques such as immunoblotting, immunofluorescence, immunoprecipitation, and ChIP assays .

• Chip peptide array technology - This approach can be used to identify phosphoacceptor consensus sites by screening kinase superfamily members against arrays containing naturally occurring phosphoacceptor sites .

• In vitro kinase screens - These help identify novel phosphoacceptor sites, as demonstrated in the discovery of the Ser269 phosphorylation site in the p53 DNA-binding domain .

• Luciferase reporter assays - These assess the transcriptional activity of wild-type versus phosphomimetic or phospho-null mutants of p53, helping to determine the functional consequences of phosphorylation events .

• Paramagnetic relaxation enhancement (PRE) experiments - These sophisticated NMR approaches can detect changes in protein conformation and interactions upon phosphorylation .

• Fluorescence anisotropy assays - These techniques can quantitatively measure changes in DNA binding capacity of p53 following phosphorylation events .

Each methodology offers distinct advantages and should be selected based on the specific research question being addressed.

How can researchers verify the specificity of phospho-specific antibodies?

Verifying antibody specificity is crucial for obtaining reliable experimental results. Based on approaches described for phospho-specific antibodies, researchers should implement multiple validation strategies:

• Phosphatase treatment controls - Compare antibody binding between phosphorylated samples and samples treated with phosphatases. A genuine phospho-specific antibody will show significantly reduced or abolished binding after phosphatase treatment .

• Mutational analysis - Test antibody reactivity against phosphomimetic mutants (e.g., S→D or S→E substitutions) versus non-phosphorylatable alanine mutants (S→A). A true phospho-specific antibody should show differential binding patterns .

• Induction experiments - Verify that antibody reactivity increases following treatments known to induce the specific phosphorylation event (e.g., DNA damage for many p53 phosphorylation sites) .

• Peptide competition assays - Perform blocking experiments with phosphorylated versus non-phosphorylated peptides to confirm binding specificity.

For example, phospho-Ser269-specific monoclonal antibodies were generated and verified by demonstrating that p53 phosphorylation is induced at Ser269 after irradiation with kinetics similar to those of p53 protein induction .

How does phosphorylation affect the structural properties of p53?

Phosphorylation events can profoundly impact the structural properties of p53, particularly affecting the dynamic equilibrium between different conformational states. For instance, phosphorylation at Ser269 was found to modulate the dynamic equilibrium between native and unfolded states of wild-type p53, specifically affecting the conformationally flexible multiprotein binding site in the p53 DNA-binding domain .

Similarly, phosphorylation at Thr55 enhances intramolecular interactions between specific domains of p53. NMR studies have revealed that Thr55 phosphorylation enhances the AD2–DBD (second activation domain–DNA-binding domain) interaction at the expense of the AD1–DBD interaction . The strength of these interactions is fine-tuned by the intervening proline-rich domain (PRD) .

These structural modifications directly translate to functional consequences, as demonstrated by the observation that phosphorylation can result in autoinhibition of p53 binding to promoters that regulate cell cycle arrest and apoptosis .

What is the relationship between p53 phosphorylation and its DNA-binding properties?

Phosphorylation plays a crucial role in regulating p53's DNA-binding capabilities, thereby controlling its transcriptional activity. Research has demonstrated that phosphorylation of p53 at Thr55 leads to dissociation of p53 from DNA at physiologically relevant concentrations .

Fluorescence anisotropy experiments with preformed p53:DNA complexes revealed that upon phosphorylation at Thr55, there was a substantial reduction in fluorescence anisotropy, indicating complete dissociation from DINP1 DNA and partial dissociation from the p21 consensus site . This effect is specific to Thr55 phosphorylation, as control experiments with p53 constructs lacking this site showed no change in DNA binding upon treatment with the same kinase .

The binding dynamics can be quantified as shown in the following table from the research studies:

These data demonstrate that phosphorylation increases the dissociation constant (Kd) for DNA binding, indicating reduced affinity .

How do multiple phosphorylation events interact to regulate p53 function?

Multiple phosphorylation events can act synergistically to fine-tune p53 function. For example, simultaneous phosphorylation of both S46 and T55 causes larger chemical shift perturbations for the N-terminal AD2 residues close to S46 than observed with T55 phosphorylation alone .

The combined effect of these phosphorylation events reduces the average height of the AD2 and PRD amide cross-peaks to a far greater extent than observed upon phosphorylation of T55 alone, indicating stronger domain interactions . Additionally, the W53 NεH cross-peak is shifted further upon dual phosphorylation and is further reduced in height .

These synergistic effects translate to functional consequences as demonstrated by the following binding parameters:

This table clearly demonstrates that dual phosphorylation at S46 and T55 results in a much more dramatic reduction in DNA binding affinity than phosphorylation at T55 alone .

What controls should be included when using phospho-specific antibodies?

When designing experiments with phospho-specific antibodies like the Phospho-TP53 (S9) antibody, researchers should implement rigorous controls to ensure valid and interpretable results:

• Phosphatase treatment controls - Samples treated with lambda phosphatase or other appropriate phosphatases serve as negative controls and should show significantly reduced or eliminated signal with a truly phospho-specific antibody .

• Kinase treatment controls - Where applicable, in vitro kinase treatment of samples can serve as positive controls by enhancing the relevant phosphorylation signal.

• Phosphomimetic and phospho-null mutants - When studying cellular systems, comparing wild-type p53 with phosphomimetic (S→D or S→E) and non-phosphorylatable (S→A) mutants can provide important insights into antibody specificity and phosphorylation function .

• Induction experiments - Testing antibody reactivity following treatments known to induce or reduce the specific phosphorylation (e.g., DNA damaging agents for many p53 phosphorylation sites) .

• Cross-reactivity controls - Testing the antibody against related phosphorylation sites or phospho-epitopes to ensure signal specificity.

For example, in studies of Ser269 phosphorylation, researchers demonstrated that phospho-Ser269-specific monoclonal antibodies showed increased reactivity after irradiation with kinetics that paralleled p53 protein induction, confirming both specificity and biological relevance .

How can researchers address discrepancies in phosphorylation-related experimental results?

Phosphorylation studies can sometimes yield conflicting results due to various experimental factors. When facing discrepancies, researchers should consider:

• Cell type and context dependencies - Different cell lines may show varying patterns of p53 phosphorylation due to differences in kinase/phosphatase activities or other regulatory mechanisms .

• Stimulus specificity - Different stress stimuli (UV, ionizing radiation, chemical agents) may induce distinct phosphorylation patterns .

• Temporal dynamics - The timing of sample collection is critical as phosphorylation events are often transient and follow specific kinetics after stimulation .

• Antibody characteristics - Different antibodies targeting the same phosphorylation site may have varying affinities, epitope specificities, or cross-reactivities .

• Detection method limitations - Different techniques (western blotting, immunofluorescence, mass spectrometry) have varying sensitivities and may yield different results .

• Extraction conditions - Phosphorylation status can be affected by sample preparation methods, with some phospho-epitopes being particularly labile during processing.

To address these issues, researchers should employ multiple complementary techniques, utilize several antibodies when available, and carefully control experimental conditions including cell density, passage number, and treatment protocols.

How does p53 phosphorylation at different sites contribute to cancer development?

The relationship between p53 phosphorylation and cancer development is complex and site-specific. Dysregulation of p53 phosphorylation can lead to uncontrolled cell proliferation and is often observed in cancer cells . The specific mechanisms vary by phosphorylation site:

For Ser269 phosphorylation, research has shown that phosphomimetic mutation at this site inactivates the transcription activation function and clonogenic suppressor activity of p53 . This suggests that hyperphosphorylation at Ser269 could contribute to cancer development by impairing p53's tumor suppressor functions.

Similarly, the phosphorylation status of Thr55 controls both activation and termination of p53-mediated transcriptional programs during different stages of the cellular DNA damage response . Dysregulation of this switch could therefore disrupt normal cellular responses to DNA damage, potentially contributing to genomic instability and cancer development.

These findings highlight the importance of precisely regulated phosphorylation in maintaining p53's tumor suppressor functions and suggest that targeting specific phosphorylation events could have therapeutic potential.

What are the future research directions for Phospho-TP53 (S9) antibody applications?

Several promising research directions emerge for applications of the Phospho-TP53 (S9) antibody:

These research directions could significantly advance our understanding of p53 regulation and potentially lead to new therapeutic strategies targeting this critical tumor suppressor pathway.