PPME1 Antibody
Description
Definition and Biological Function of PPME1
PPME1 (Protein Phosphatase Methylesterase-1) is a conserved serine hydrolase that demethylates the PP2A catalytic subunit (PP2Ac) at leucine 309, modulating PP2A’s activity and substrate specificity . PP2A is a tumor suppressor phosphatase involved in regulating growth, apoptosis, and signal transduction pathways like ERK and AKT . PPME1 antibodies target this enzyme for detection in experimental and clinical settings.
Research Findings in Cancer Biology
PPME1 amplification and overexpression are linked to tumor progression in gastric cancer (GC) and lung cancer (LC):
Key Observations:
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Gene Amplification: Detected in 3.8% of GC and 3.1% of LC patient samples, correlating with elevated PPME1 protein levels .
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Functional Impact:
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Clinical Relevance: PPME1 immunopositivity correlates with higher Ki-67 proliferation indices in glioblastomas, indicating a role in malignant progression .
Mechanistic Insights from Knockout Studies
PME-1 knockout mouse embryonic fibroblasts (MEFs) revealed:
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Reduced PP2A Stability: PP2Ac protein levels decreased by 60–65% in PME-1 KO cells due to enhanced ubiquitin/proteasome degradation .
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Paradoxical Activity Loss: Despite PME-1’s role as a PP2A inhibitor, its knockout reduced PP2A activity by 30–50%, likely due to lower PP2Ac abundance .
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Methylation-Dependent Protection: PME-1’s methylesterase activity stabilizes PP2Ac, as shown by cycloheximide chase assays (35% PP2Ac remaining in KO vs. 70% in WT after 24 hours) .
Implications for Therapeutic Development
PPME1 is emerging as a cancer therapeutic target:
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Amplification-Driven Dependency: PPME1-amplified cancer cells show heightened sensitivity to PPME1 inhibition, unlike non-amplified cells .
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Drug Development: Selective small-molecule inhibitors of PPME1 are under investigation, leveraging its low expression in normal tissues (except brain) for therapeutic windows .
Product Specs
Q&A
How should researchers validate PPME1 antibody specificity for Western blotting?
Validation requires a multi-step approach:
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Knockout/Knockdown Controls: Use siRNA or CRISPR-Cas9 to reduce PPME1 expression in cell lysates. Compare bands in treated vs. untreated samples at ~42 kDa (expected molecular weight) . Search result demonstrates nonspecific binding of PP2A-related antibodies without proper controls, emphasizing the need for genetic validation.
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Peptide Blocking: Pre-incubate the antibody with immunogenic peptides (e.g., residues 1–386 of human PPME1 ). A ≥80% reduction in band intensity confirms specificity.
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Cross-Reactivity Profiling: Test antibody reactivity across species (human, mouse, rat) using recombinant proteins. Clone 8A6-F8 shows broad reactivity , whereas PAT29G3AT may require optimization .
Table 1: Validation Parameters for Common PPME1 Antibodies
| Clone | Host | Applications Validated | Recommended Dilution | Key Controls |
|---|---|---|---|---|
| 8A6-F8 | Mouse | WB, IHC-P | 1:1,000 (WB) | KO lysates |
| PAT29G3AT | Mouse | WB, ELISA | 1:500 (WB) | Peptide blocking |
What methodological considerations optimize PPME1 immunohistochemistry (IHC) in formalin-fixed tissues?
Optimal IHC requires:
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Antigen Retrieval: Use citrate buffer (pH 6.0) at 95°C for 20 minutes to expose epitopes masked by formalin .
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Antibody Titration: Test concentrations between 2–10 µg/mL. Overexpression in hepatocellular carcinoma (HCC) tissues permits lower concentrations .
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Signal Amplification: Employ horseradish peroxidase (HRP)-polymer systems to enhance sensitivity for low-abundance targets.
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Quantification: Use H-scores incorporating staining intensity (0–3) and percentage of positive cells. Studies correlate H-scores >200 with poor HCC prognosis .
How do storage conditions affect PPME1 antibody performance?
PPME1 antibodies without preservatives (e.g., sodium azide) require aseptic aliquoting to prevent microbial growth. Long-term storage at -20°C in 50% glycerol prevents freeze-thaw damage . Post-reconstitution stability varies:
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Cell Signaling Technology’s 8A6-F8: Stable for 6 months at 4°C .
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Prospec’s PAT29G3AT: Degrades after 1 month at 4°C; requires -20°C storage .
How can contradictory data on PPME1’s role in PP2A regulation be resolved?
Discrepancies arise from:
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Antibody Cross-Reactivity: Some antibodies recognize both methylated and unmethylated PP2A-C Leu309 . Combine methylation-sensitive antibodies (e.g., Millipore 07-468) with PP2A activity assays to disentangle effects .
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Cell Line-Specific Effects: PPME1 knockdown inhibits proliferation in SNU668 (gastric) and Oka-C1 (lung) cells but not in MKN1 or HCC95 . Validate findings across ≥3 models with confirmed PPME1 amplification via FISH .
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Post-Translational Modifications: PP2A-C phosphorylation at Tyr307 alters antibody binding . Treat lysates with λ-phosphatase to eliminate phosphorylation confounders.
What functional assays best characterize PPME1’s oncogenic activity?
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Demethylation Activity Assays:
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Pathway Modulation:
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In Vivo Tumorigenesis:
How does PPME1 interact with epigenetic regulators in cancer?
PPME1 overexpression correlates with DNA hypermethylation in CpG islands of tumor suppressors:
Product Science Overview
Mouse anti-human antibodies are antibodies produced in mice that are specific to human antigens. These antibodies are commonly used in research and diagnostic applications due to their specificity and affinity for human proteins .
Production and Applications
Mouse anti-human antibodies are generated by immunizing mice with human antigens. The immune response in mice leads to the production of antibodies that can be harvested and purified for various applications. These antibodies are widely used in immunoassays, flow cytometry, and other techniques to detect and quantify human proteins .
Human Anti-Mouse Antibody (HAMA) Response
One challenge associated with the use of mouse anti-human antibodies is the potential for the human anti-mouse antibody (HAMA) response. HAMA is an immune response in humans against mouse-derived antibodies, which can lead to reduced efficacy and adverse reactions . To mitigate this, researchers have developed humanized and fully human antibodies that minimize the risk of HAMA .
