PES1 Antibody

What is PES1 and why is it significant in research?

PES1 (pescadillo homolog 1, containing BRCT domain) is a nuclear protein involved in ribosome biogenesis and is associated with cellular proliferation and tumorigenesis. It contains a breast cancer associated gene 1 (BRCA1) C-terminal interaction domain and has been implicated in various cancer types, making it an important target for cancer research . The protein has a calculated molecular weight of 68 kDa (588 amino acids) but is typically observed at 72 kDa in experimental conditions . Its involvement in essential cellular processes makes PES1 a promising target for understanding cancer development and identifying potential therapeutic interventions . Recent studies have shown that PES1 overexpression promotes carcinogenesis in multiple malignant tumors, including pancreatic cancer, where it contributes to poor prognosis .

What applications are PES1 antibodies validated for in research?

PES1 antibodies have been validated for multiple research applications, with specific reactivity profiles depending on the antibody clone. For example, the 13553-1-AP antibody has been validated for Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Immunoprecipitation (IP), and ELISA applications . The recommended dilutions vary by application:

It's important to note that these dilutions should be optimized for each specific experimental system to obtain optimal results, as they can be sample-dependent . Similarly, other PES1 antibodies like CAB14506 have been specifically validated for WB and ELISA applications with a recommended WB dilution range of 1:500-1:2000 .

How should PES1 antibodies be stored and handled for optimal performance?

For optimal performance, PES1 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage, which simplifies handling procedures. Some preparations (particularly 20µl sizes) may contain 0.1% BSA as a stabilizer . When working with the antibody, it's advisable to thaw it completely and maintain it on ice during use. Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody and subsequent loss of binding efficiency.

What are the best practices for using PES1 antibodies in immunohistochemistry (IHC)?

When performing immunohistochemistry with PES1 antibodies, several methodological considerations are critical for obtaining reliable results. For antigen retrieval, it is recommended to use TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . Human gliomas tissue has been verified as positive control material for IHC applications with PES1 antibodies .

For scoring IHC results, a standardized approach involves calculating a score as the product of staining intensity and the proportion of positive tumor cells. The staining intensity should be graded according to specific criteria: 1 = weak staining at 100× magnification but little or no staining at 40× magnification; 2 = medium staining at 40× magnification; 3 = strong staining at 40× magnification . This scoring system was effectively used in research examining PES1 expression in pancreatic cancer, where independent assessment by two experienced pathologists who were blinded to patient information ensured objectivity . For antibody working dilutions, testing has shown that 1:50-1:500 typically provides optimal results for IHC applications , though specific optimization may be necessary for different tissue types or experimental questions.

How can researchers optimize Western blot protocols for PES1 detection?

For optimal detection of PES1 via Western blot, researchers should consider several technical factors. The antibody dilution range of 1:2000-1:10000 has been validated for PES1 detection , with some antibodies like CAB14506 performing optimally in the 1:500-1:2000 range . When selecting positive controls, multiple cell lines have been validated, including COLO 320 cells and MCF-7 cells for Western blot applications , as well as MCF7, HepG2, and Jurkat cells for certain antibody clones .

The expected molecular weight of PES1 is approximately 68 kDa (calculated), but the observed molecular weight in experimental conditions is typically 72 kDa . This discrepancy should be considered when interpreting Western blot results. Researchers should follow antibody-specific Western blot protocols which may be available from manufacturers . For reproducible results, standardization of protein loading (15-30 µg of total protein per lane), appropriate blocking (typically 5% non-fat milk or BSA), and overnight primary antibody incubation at 4°C are recommended practices. Additionally, researchers should be aware that post-translational modifications of PES1, such as phosphorylation by CDK5, can affect protein stability and potentially its migration pattern on gels .

What controls should be included when working with PES1 antibodies?

Proper controls are essential for validating results obtained with PES1 antibodies. For positive controls in Western blot applications, COLO 320 cells and MCF-7 cells have been validated , while MCF7, HepG2, and Jurkat cells are recommended for certain antibody clones . For immunoprecipitation, COLO 320 cells serve as a validated positive control . In immunohistochemistry, human gliomas tissue has been verified for PES1 antibody applications , and for immunofluorescence/ICC, MCF-7 cells are recommended .

Negative controls should include samples where PES1 is known to be absent or knockdown/knockout models. In research studying PES1 function, shRNA-mediated knockdown models have been successfully employed to validate antibody specificity and functional outcomes . Additionally, isotype controls (using non-specific rabbit IgG at the same concentration as the primary antibody) should be included to assess non-specific binding. When performing functional studies, both gain-of-function (overexpression) and loss-of-function (knockdown) approaches should be considered, as demonstrated in studies examining PES1's role in drug sensitivity in pancreatic cancer cells .

How can researchers assess PES1 involvement in protein-protein interactions?

To investigate PES1's protein-protein interactions, immunoprecipitation (IP) followed by Western blot analysis (co-IP) is the most commonly employed technique. For IP applications, it is recommended to use 0.5-4.0 µg of antibody for 1.0-3.0 mg of total protein lysate . This approach has been successfully used to demonstrate PES1's interaction with BRD4 in pancreatic cancer cells, revealing its role in enhancing c-Myc expression .

For more comprehensive analysis of PES1 interactomes, researchers can combine IP with mass spectrometry. When designing these experiments, it's crucial to include appropriate controls such as IgG controls and input samples. Crosslinking prior to IP may be beneficial for capturing transient interactions. Alternative approaches include proximity-based labeling methods such as BioID or APEX, which can identify proteins in close proximity to PES1 in living cells. Additionally, yeast two-hybrid screens can be employed for initial identification of potential interacting partners, though results should be validated using co-IP or other methods in mammalian cells. When reporting interaction data, researchers should include quantitative assessments of binding strength and specificity alongside biological replicates to ensure reproducibility .

What are the challenges in studying PES1 phosphorylation and how can they be addressed?

Studying PES1 phosphorylation presents several challenges that require specific methodological approaches. Research has shown that PES1 is phosphorylated and stabilized by CDK5 in pancreatic cancer cells, which affects its function . To effectively study this post-translational modification, researchers should consider phospho-specific antibodies when available, or use general phospho-detection methods such as Phos-tag gels which can separate phosphorylated from non-phosphorylated forms of the protein.

Mass spectrometry approaches are valuable for identifying specific phosphorylation sites on PES1. Sample preparation for such analysis should include phosphatase inhibitors in all buffers to prevent dephosphorylation during extraction. To evaluate the functional significance of phosphorylation, site-directed mutagenesis can be employed to create phosphomimetic (e.g., serine to aspartate) or phospho-deficient (e.g., serine to alanine) mutants of PES1. Kinase inhibitor studies, such as those using the CDK5 inhibitor Dinaciclib, can help validate kinase-substrate relationships in vitro and in vivo . Researchers should also consider the dynamic nature of phosphorylation by performing time-course experiments following stimulation or inhibition of relevant signaling pathways. The integration of these approaches provides a comprehensive understanding of how phosphorylation regulates PES1 stability and function in various cellular contexts.

What strategies can resolve antibody cross-reactivity or non-specific binding issues with PES1 antibodies?

Cross-reactivity and non-specific binding can compromise the reliability of PES1 antibody-based experiments. To address these issues, researchers should first validate antibody specificity using PES1 knockdown or knockout controls. Several published studies have successfully utilized shRNA-mediated knockdown of PES1 to confirm antibody specificity . Additionally, researchers should be aware of the exact epitope recognized by their specific antibody clone. For instance, some antibodies are generated against PES1 fusion protein Ag4342, while others target specific amino acid sequences (e.g., amino acids 1-150 of human PES1) .

Optimizing blocking conditions is crucial—typically using 5% non-fat milk or BSA in TBS-T for Western blot or 5-10% normal serum from the same species as the secondary antibody for IHC/IF applications. Titrating antibody concentrations is essential, with recommended dilution ranges varying by application (1:2000-1:10000 for WB, 1:50-1:500 for IHC/IF) . Pre-absorption of the antibody with the immunizing peptide (when available) can help confirm specificity. For IHC applications, antigen retrieval optimization is important, with recommendations suggesting TE buffer at pH 9.0 or alternative citrate buffer at pH 6.0 . Finally, reducing secondary antibody concentration or switching to highly cross-adsorbed secondary antibodies can minimize background in multichannel fluorescence applications.

How does PES1 expression correlate with cancer prognosis and how can antibodies be used to assess this?

PES1 expression has been established as a prognostic biomarker in several cancer types, with its overexpression generally correlating with poor patient outcomes. Research using PES1 antibodies in tissue microarray (TMA) analysis has revealed that PES1 is abnormally increased in pancreatic cancer tissues and correlates with poor prognosis for these patients . For prognostic studies, immunohistochemistry (IHC) using validated PES1 antibodies (recommended dilution 1:50-1:500) is the preferred method for analyzing expression in patient samples .

The scoring system for IHC evaluation should be standardized, with scores calculated as the product of staining intensity (graded 1-3) and the proportion of positive tumor cells . This scoring approach enables reliable stratification of patients into high and low PES1 expression groups for survival analysis. Researchers should ensure blinded assessment by at least two independent pathologists to minimize bias . Complementary methods such as RT-qPCR can validate IHC findings at the mRNA level. For large-scale prognostic studies, automated image analysis of IHC staining can provide more objective quantification. Additionally, multiplexed IHC including PES1 alongside other markers (such as c-Myc) can provide insights into functional relationships and improved prognostic value . These methodological considerations are crucial for reliable assessment of PES1's prognostic significance across different cancer types.

What is the role of PES1 in drug resistance and how can researchers investigate this?

PES1 has been identified as a significant contributor to drug resistance in cancer, particularly to BET inhibitors in pancreatic cancer. Research has demonstrated that PES1 interacts with BRD4 to enhance c-Myc expression, which is a primary cause of resistance to BET inhibitors . To investigate PES1's role in drug resistance, researchers can employ several methodological approaches.

Cell viability assays (such as MTS) comparing drug sensitivity between PES1 knockdown, overexpression, and control cells provide fundamental insights. In pancreatic cancer research, this approach revealed that knockdown of PES1 increased sensitivity to BET inhibitors (JQ1), while PES1 overexpression promoted resistance . The experimental design should include determination of IC50 values across a range of drug concentrations, as demonstrated in the following findings:

Mechanistic studies should investigate protein-protein interactions (such as PES1-BRD4) using co-immunoprecipitation, and downstream effects on target genes like c-Myc using Western blot and qPCR . Combination treatment approaches, such as CDK5 inhibitors (Dinaciclib) with BET inhibitors (JQ1), can be evaluated to overcome PES1-mediated resistance both in vitro and in vivo using xenograft models . These methodological approaches provide a comprehensive framework for investigating PES1's role in drug resistance across various cancer types and therapeutic contexts.

How can researchers use PES1 antibodies to study its interaction with BRD4 and effects on c-Myc expression?

Investigating the PES1-BRD4-c-Myc axis requires a multi-faceted experimental approach centered around various antibody-based techniques. Co-immunoprecipitation (co-IP) using PES1 antibodies (recommended 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) followed by BRD4 detection, or vice versa, can confirm direct interaction between these proteins . This approach has successfully demonstrated the PES1-BRD4 interaction in pancreatic cancer cells .

For analyzing downstream effects on c-Myc expression, comparative Western blot analysis in PES1 knockdown, overexpression, and control conditions provides quantitative assessment of protein level changes. Immunofluorescence co-localization studies using antibodies against PES1, BRD4, and c-Myc can reveal spatial relationships within the nucleus, particularly in the nucleolus where PES1 is predominantly localized . Chromatin immunoprecipitation (ChIP) assays using PES1 and BRD4 antibodies can determine co-occupancy at c-Myc promoter regions, elucidating the transcriptional regulatory mechanism.

To establish the functional significance of this interaction, researchers should perform rescue experiments where c-Myc is exogenously expressed in PES1-knockdown cells to determine if it restores the original phenotype. For translational relevance, correlative IHC studies on patient tissues can assess the relationship between PES1 and c-Myc expression patterns . When combined, these methodological approaches provide comprehensive insights into how PES1 regulates c-Myc expression through interaction with BRD4, and how this mechanism contributes to cancer progression and drug resistance.

How can PES1 antibodies be used to study ribosome biogenesis in cancer cells?

PES1's role in ribosome biogenesis represents a critical area for cancer research, as dysregulated ribosome production is a hallmark of many malignancies. To study this function, researchers can employ PES1 antibodies in several advanced applications. Nucleolar co-localization studies using immunofluorescence (recommended dilution 1:50-1:500) with PES1 antibodies alongside markers for different nucleolar compartments can reveal the spatial distribution of PES1 during ribosome assembly . This approach is facilitated by PES1's known cellular localization in the chromosome, nucleus, nucleolus, and nucleoplasm .

For functional studies, researchers should combine PES1 knockdown or overexpression with analyses of pre-rRNA processing using Northern blotting or qRT-PCR for specific precursor rRNAs. Polysome profiling followed by Western blot detection of PES1 in different fractions can identify its association with specific ribosomal assembly intermediates. Proximity labeling methods (BioID, APEX) using PES1 as bait can identify novel interacting partners in the ribosome biogenesis pathway.

To connect ribosome biogenesis defects with cancer phenotypes, researchers should assess how PES1 manipulation affects global protein synthesis rates (using puromycin incorporation or metabolic labeling) and the translation of specific oncogenic mRNAs. The relationship between PES1-mediated ribosome biogenesis and cancer cell response to nucleolar stress (induced by agents like actinomycin D) can provide insights into potential therapeutic vulnerabilities. These methodological approaches collectively enable comprehensive investigation of PES1's role in the dysregulated ribosome biogenesis that supports cancer cell growth and survival.

What are the considerations for developing phospho-specific PES1 antibodies for cancer research?

Developing phospho-specific PES1 antibodies represents an important frontier in cancer research, particularly given the finding that CDK5 phosphorylates and stabilizes PES1 in pancreatic cancer cells . For researchers considering this approach, several methodological considerations are essential. First, identification of the precise phosphorylation sites is critical—this typically requires mass spectrometry analysis of PES1 immunoprecipitated from cancer cells, with and without CDK5 inhibitor treatment .

Once phosphorylation sites are identified, phospho-peptides representing these sites should be synthesized for antibody production. These peptides should be 10-15 amino acids in length with the phosphorylated residue centrally positioned, and conjugated to a carrier protein (typically KLH). For antibody generation, a two-rabbit immunization protocol is recommended, with ELISA-based screening against both phosphorylated and non-phosphorylated peptides to select clones with at least 100-fold specificity for the phosphorylated form.

Rigorous validation is crucial and should include: (1) Western blot comparison of signals from wild-type cells versus those treated with phosphatase or relevant kinase inhibitors like Dinaciclib ; (2) testing against phospho-deficient PES1 mutants (e.g., serine to alanine); (3) peptide competition assays to confirm specificity; and (4) immunoprecipitation followed by mass spectrometry to confirm the detected protein is indeed phosphorylated PES1. Applications of these phospho-specific antibodies would enable researchers to monitor PES1 phosphorylation dynamics in response to therapeutic interventions, potentially serving as pharmacodynamic biomarkers for CDK5 inhibitor efficacy in cancer treatment .

How can multiplexed imaging approaches with PES1 antibodies advance cancer research?

Multiplexed imaging incorporating PES1 antibodies offers powerful opportunities to advance cancer research by simultaneously visualizing multiple molecular markers within the same tissue section or cell. For effective multiplexed immunofluorescence including PES1, researchers should consider several methodological approaches. Spectral unmixing systems can accommodate 4-8 fluorophores in a single experiment, allowing co-detection of PES1 (typically using rabbit polyclonal antibodies at 1:50-1:500 dilution) alongside markers of cell proliferation, apoptosis, and cancer-specific pathways.

Cyclic immunofluorescence methods, which involve iterative staining-imaging-bleaching cycles, can extend multiplexing capability to 20+ markers on the same sample. This approach would allow comprehensive characterization of the PES1 microenvironment in tumors. Mass cytometry imaging (e.g., MIBI, IMC) using metal-conjugated PES1 antibodies can achieve 40+ marker detection without spectral overlap constraints, though specialized equipment is required.

These approaches enable several advanced research applications: (1) correlation of PES1 expression with cellular states (proliferation/differentiation/stemness) across heterogeneous tumor regions; (2) simultaneous visualization of the entire PES1-BRD4-c-Myc axis within individual cells ; (3) assessment of PES1 expression in specific immune cell populations within the tumor microenvironment; and (4) evaluation of PES1 as a response biomarker in pre- and post-treatment samples. For analyzing the resulting complex datasets, machine learning approaches can identify novel cell populations and spatial relationships not apparent through conventional single-marker approaches, potentially revealing new insights into PES1's role in cancer biology.