PPM1L Antibody

What is PPM1L and what are its primary cellular functions?

PPM1L, also known as protein phosphatase 1-like or PP2C-epsilon, is a Mg²⁺/Mn²⁺-dependent serine/threonine phosphatase that acts as a suppressor of the SAPK signaling pathways. It functions by associating with and dephosphorylating MAP3K7/TAK1 and MAP3K5 . PPM1L is essential for maintaining genomic stability and controlling cell growth and division . It is involved in the H₂O₂-induced regulation of ASK1 signaling and participates in the regulation of TGF-β and BMP (Bone Morphogenetic Protein) signaling pathways . Dysregulation of PPM1L has been linked to cancer development and progression, making it a promising target for cancer research .

What applications are PPM1L antibodies typically used for?

PPM1L antibodies are validated for multiple research applications:

These applications enable researchers to study PPM1L expression patterns, protein interactions, and functional roles in various experimental systems .

What species reactivity can I expect from commercially available PPM1L antibodies?

Most commercial PPM1L antibodies demonstrate reactivity with human and mouse samples . Some antibodies have also been validated for rat samples . When selecting an antibody for your research, verify the species reactivity in the product documentation. For example, the polyclonal antibody catalog number 18203-1-AP has confirmed reactivity with human, mouse, and rat samples , while other antibodies like PACO22527 specifically react with human and mouse samples .

How should PPM1L antibodies be stored to maintain their efficacy?

For optimal stability and performance, PPM1L antibodies should be stored at -20°C . Most commercial preparations remain stable for one year after shipment when properly stored . The antibodies are typically supplied in storage buffers containing either:

• PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 , or

• Phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide and 50% glycerol

What controls should I include when using PPM1L antibodies in Western blotting?

When designing Western blot experiments with PPM1L antibodies, incorporate these essential controls:

• Positive tissue control: Mouse kidney tissue has been validated as a positive control for PPM1L expression . Jurkat cell extracts have also been used successfully to detect PPM1L .

• Negative control: Include samples where PPM1L expression is known to be absent or significantly reduced.

• Loading control: Use housekeeping proteins (β-actin, GAPDH, tubulin) to normalize PPM1L expression.

• Molecular weight markers: PPM1L has a calculated molecular weight of 26 kDa and 41 kDa, but is typically observed at 41-45 kDa in Western blots . This information is critical for proper band identification.

• Antibody specificity control: Consider using siRNA-mediated knockdown of PPM1L or recombinant PPM1L protein as additional specificity controls.

When analyzing results, be aware that PPM1L has 4 isoforms produced by alternative splicing , which might appear as multiple bands in some tissue types.

What is the optimal protocol for immunohistochemical detection of PPM1L?

For successful immunohistochemical (IHC) detection of PPM1L in tissue samples:

• Sample preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections.

• Antigen retrieval: Perform antigen retrieval using TE buffer pH 9.0 (preferred) or alternatively citrate buffer pH 6.0 .

• Blocking: Block with appropriate serum (typically 5-10% normal goat serum) to reduce background staining.

• Primary antibody incubation: Apply PPM1L antibody at a dilution of 1:20-1:200 and incubate at 4°C overnight.

• Detection system: Use a compatible detection system based on your primary antibody (e.g., HRP-conjugated secondary antibody).

• Positive control tissues: Human kidney and heart tissues have been validated as positive controls for PPM1L IHC staining .

• Visualization: Develop with an appropriate chromogen (typically DAB) and counterstain with hematoxylin.

The staining pattern should be evaluated against known expression patterns, with particular attention to subcellular localization that corresponds to PPM1L's biological functions.

How can I optimize Western blot conditions for detecting PPM1L?

To achieve optimal Western blot results when detecting PPM1L:

• Sample preparation: Use RIPA or NP-40 lysis buffers with protease and phosphatase inhibitors. Jurkat cells have been successfully used for PPM1L detection .

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution of PPM1L (observed MW: 41-45 kDa) .

• Transfer conditions: Transfer to PVDF membrane at 100V for 60-90 minutes in standard transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol).

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary PPM1L antibody 1:500-1:1000 in blocking buffer and incubate overnight at 4°C.

• Washing and detection: Wash thoroughly with TBST and use HRP-conjugated secondary antibody with appropriate chemiluminescent detection system.

• Exposure time optimization: Start with 30-second exposure and adjust as needed based on signal intensity.

If non-specific bands appear, consider increasing washing stringency or further diluting the primary antibody. Remember that PPM1L has multiple isoforms that may appear as distinct bands .

How can PPM1L antibodies be used to investigate its role in cancer progression?

PPM1L dysregulation has been linked to cancer development and progression . Researchers can leverage PPM1L antibodies to investigate this connection through multiple approaches:

• Expression profiling: Compare PPM1L expression levels between normal and cancerous tissues using IHC and Western blot analysis. Human tissue microarrays can be employed to assess expression across multiple cancer types simultaneously.

• Correlation studies: Analyze the relationship between PPM1L expression levels and clinical outcomes, tumor grade, or metastatic potential.

• Mechanistic investigations: Use PPM1L antibodies in co-immunoprecipitation experiments to identify novel interaction partners in cancer cells, potentially revealing altered signaling pathways.

• Phosphorylation state analysis: Combine PPM1L antibodies with phospho-specific antibodies to examine how PPM1L activity affects the phosphorylation status of its substrates in cancer cells.

• Therapeutic response monitoring: Evaluate changes in PPM1L expression or activity following treatment with anti-cancer agents to determine its potential as a biomarker for treatment response.

Research has indicated that PPM1L may be particularly relevant in colorectal cancer, as genome-wide scans have identified a copy number variable region at 3q26 that regulates PPM1L in APC mutation-negative familial colorectal cancer patients .

What are the technical considerations for using PPM1L antibodies in co-immunoprecipitation experiments?

When using PPM1L antibodies for co-immunoprecipitation (co-IP) to study protein-protein interactions:

• Antibody selection: Choose a PPM1L antibody specifically validated for immunoprecipitation applications. Polyclonal antibodies often perform better in co-IP compared to monoclonal antibodies.

• Buffer optimization: Use a gentle lysis buffer (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.4) to preserve protein-protein interactions. Include protease and phosphatase inhibitors.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody binding conditions: For PPM1L, which functions in signaling pathways involving MAP3K7/TAK1 and MAP3K5 , incubate antibody with lysate overnight at 4°C to capture transient interactions.

• Washing stringency: Use low-stringency washing buffers initially (e.g., lysis buffer without detergent) and increase stringency gradually to maintain specific interactions while reducing background.

• Elution and detection: Elute with SDS sample buffer and analyze by Western blot using antibodies against suspected interaction partners.

• Reverse co-IP validation: Confirm interactions by performing reverse co-IP using antibodies against the interacting protein partner.

This approach has been valuable in establishing PPM1L's role in ASK1 signaling regulation and its interactions with MAP3K proteins .

How can I use PPM1L antibodies to study its role in stress response pathways?

PPM1L plays a crucial role in stress response mechanisms, particularly through regulating the SAPK signaling pathways and ASK1 activation . To investigate these functions:

• Stress induction experiments: Expose cells to stressors such as H₂O₂, TNFα, or UV radiation, then analyze PPM1L levels and localization using immunofluorescence and Western blotting.

• Kinetics studies: Perform time-course experiments following stress induction to determine the temporal relationship between PPM1L activity and stress response signaling.

• Phosphorylation analysis: Use PPM1L antibodies in conjunction with phospho-specific antibodies targeting ASK1, MAP3K7/TAK1, and downstream MAPKs to examine how PPM1L regulates phosphorylation cascades.

• Subcellular fractionation: Combine with immunoblotting to determine whether stress induces translocation of PPM1L between cellular compartments.

• Proximity ligation assay: Use PPM1L antibodies together with antibodies against interaction partners to visualize and quantify protein-protein interactions in situ following stress.

Research has demonstrated that PPM1L suppresses gene expression of SAPK by associating with and dephosphorylating MAP3K7/TAK1 and MAP3K5 . After exposure to H₂O₂, PPM1L plays a role in regulating ASK1 signaling, a pathway that can lead to apoptosis when activated .

How can I address weak or absent signal when using PPM1L antibody in Western blots?

When encountering weak or absent PPM1L signal in Western blotting, consider these troubleshooting approaches:

• Antibody concentration: Increase the antibody concentration within the recommended range (1:500-1:1000) . Some applications may require concentrations at the higher end of this range.

• Protein extraction method: PPM1L may be compartmentalized or tightly associated with other proteins. Try alternative extraction methods such as RIPA buffer with increased detergent concentration or sonication steps.

• Sample selection: Confirm you're using appropriate positive control samples. Mouse kidney tissue and Jurkat cell extracts have been validated for PPM1L detection.

• Protein loading: Increase total protein loading to 50-75 μg per lane to enhance detection of less abundant proteins.

• Membrane type: Switch from nitrocellulose to PVDF membrane, which has higher protein binding capacity.

• Extended exposure time: Increase the exposure time during chemiluminescent detection.

• Enhanced detection systems: Use more sensitive detection systems such as enhanced chemiluminescence (ECL) or fluorescence-based detection methods.

• Alternative antibody: If possible, try an alternative PPM1L antibody targeting a different epitope, such as those derived from different immunogens (e.g., DLDKLQPEFMILASDGLWDAFSNEEAVRFIKERLDEPHFGAKSIVLQSFYRGCPDNITVMVVKFRNSSK versus VLCDKDGNAIPLSHDHKPYQLKE ).

What factors might cause non-specific binding when using PPM1L antibodies in immunohistochemistry?

When troubleshooting non-specific binding in PPM1L immunohistochemistry:

• Antibody dilution: Adjust the antibody dilution within the recommended range (1:20-1:200) . Start with a more dilute solution (e.g., 1:200) and increase concentration if specific signal is weak.

• Antigen retrieval optimization: Compare the effectiveness of TE buffer pH 9.0 versus citrate buffer pH 6.0 . Different antibody clones may perform better with specific retrieval methods.

• Blocking protocol: Increase blocking time (up to 2 hours) or concentration of blocking agent (up to 10% normal serum) to reduce non-specific binding.

• Secondary antibody cross-reactivity: Ensure secondary antibody is appropriate for your primary antibody species and isotype. Consider using a secondary antibody pre-adsorbed against the species of your sample.

• Endogenous peroxidase quenching: For HRP-based detection systems, ensure complete quenching of endogenous peroxidase activity using 3% H₂O₂ for 10-15 minutes.

• Endogenous biotin blocking: If using biotin-based detection, block endogenous biotin using commercial biotin blocking kits.

• Tissue fixation: Overfixation may mask epitopes. If possible, test tissues with different fixation durations.

• Background reduction: Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding.

Remember that PPM1L antibodies have been successfully validated for human kidney and heart tissues , which can serve as positive controls for optimization.

How can I distinguish between PPM1L isoforms when interpreting Western blot results?

PPM1L has 4 isoforms produced by alternative splicing , which can complicate the interpretation of Western blot results. To address this challenge:

• Expected molecular weights: PPM1L has calculated molecular weights of 26 kDa and 41 kDa, but is typically observed at 41-45 kDa in Western blots . This information can help identify specific isoforms.

• Tissue-specific expression: Different tissues may express different isoform patterns. Document the banding pattern in various tissues to create a reference profile.

• High-resolution gels: Use 10-12% acrylamide gels with extended running times to achieve better separation between closely migrating isoforms.

• 2D gel electrophoresis: Consider using 2D gel electrophoresis (isoelectric focusing followed by SDS-PAGE) to separate isoforms with similar molecular weights but different isoelectric points.

• Isoform-specific antibodies: Where available, use antibodies that specifically recognize certain isoforms based on their unique epitopes.

• Recombinant protein standards: Run recombinant proteins representing known isoforms alongside your samples as migration references.

• Validation approaches: Employ RT-PCR or qPCR with isoform-specific primers to correlate protein bands with mRNA expression of specific isoforms.

• Knockdown confirmation: Use siRNA targeting specific isoforms to confirm band identity by observing selective reduction in intensity.

This systematic approach can help create reliable isoform profiles that enhance the interpretation of PPM1L expression patterns across experimental conditions.

How does PPM1L contribute to the regulation of TGF-β and BMP signaling pathways?

PPM1L has been associated with the regulation of the TGF-β and BMP (Bone Morphogenetic Protein) signaling pathways . Researchers can investigate this regulatory role using PPM1L antibodies through:

• Pathway component phosphorylation: Use PPM1L antibodies in combination with phospho-specific antibodies against SMAD proteins to determine how PPM1L activity affects TGF-β and BMP signal transduction.

• Reporter assays: Correlate PPM1L expression levels (quantified by Western blot) with TGF-β or BMP responsive element activity in reporter assays.

• Interaction studies: Perform co-immunoprecipitation with PPM1L antibodies followed by mass spectrometry to identify novel interactions with TGF-β and BMP pathway components.

• Subcellular co-localization: Use immunofluorescence with PPM1L antibodies and antibodies against TGF-β receptors or SMADs to examine their spatial relationship during signaling events.

• Targeted phosphatase assays: Immunoprecipitate PPM1L using specific antibodies, then perform in vitro phosphatase assays with purified, phosphorylated components of the TGF-β and BMP pathways.

Understanding PPM1L's role in these pathways is particularly relevant to cancer research, as TGF-β signaling has dual roles in tumor suppression and promotion depending on cellular context and disease stage.

What is the significance of PPM1L in colorectal cancer development?

Research has identified a potential role for PPM1L in colorectal cancer development, particularly in the context of familial cases without APC mutations . To investigate this connection:

• Expression analysis: Compare PPM1L expression levels between normal colonic mucosa and colorectal cancer samples at different stages using IHC and Western blot with validated PPM1L antibodies.

• Genetic correlation studies: Analyze the relationship between the copy number variable region at 3q26 that regulates PPM1L and clinical outcomes in colorectal cancer patients.

• Functional studies: Use PPM1L antibodies to measure protein levels after experimental manipulation of PPM1L expression (overexpression or knockdown) and assess effects on colorectal cancer cell proliferation, migration, and invasion.

• Pathway analysis: Investigate how PPM1L affects the phosphorylation status of key signaling molecules in colorectal cancer cells, particularly those in the Wnt/β-catenin pathway that is frequently dysregulated in colorectal cancer.

• Biomarker potential: Evaluate PPM1L as a potential diagnostic or prognostic biomarker by correlating its expression levels with patient outcomes using tissue microarrays and PPM1L antibodies.

This research direction is supported by findings that a copy number variable region at 3q26 regulates PPM1L in APC mutation-negative familial colorectal cancer patients , suggesting PPM1L may be relevant to alternative mechanisms of colorectal cancer development.

How can PPM1L antibodies be used to study its role in H₂O₂-induced ASK1 signaling regulation?

PPM1L is involved in H₂O₂-induced regulation of ASK1 signaling, a pathway leading to apoptosis . Researchers can use PPM1L antibodies to investigate this regulatory mechanism through:

• Oxidative stress experiments: Treat cells with varying concentrations of H₂O₂, then analyze changes in PPM1L phosphorylation state, expression level, and subcellular localization using specific antibodies.

• Time-course analysis: Following H₂O₂ exposure, collect samples at multiple time points to determine the temporal relationship between PPM1L activity and ASK1 phosphorylation/activation.

• Protein complex analysis: Use PPM1L antibodies for co-immunoprecipitation experiments before and after H₂O₂ treatment to identify dynamic changes in PPM1L-containing protein complexes.

• Phosphatase activity assays: Immunoprecipitate PPM1L using specific antibodies and measure its phosphatase activity toward ASK1 under various oxidative conditions.

• Mechanistic studies: Combine PPM1L antibodies with site-specific phospho-ASK1 antibodies to determine which phosphorylation sites on ASK1 are regulated by PPM1L.

• Subcellular fractionation: Use PPM1L antibodies to track potential translocation of PPM1L between cellular compartments during oxidative stress response.

This approach builds on research showing that ASK1 is activated in response to cytotoxic stresses such as H₂O₂ and TNFα, initiating a signaling cascade leading to apoptosis, with PPM1L playing a regulatory role in this process .

How does PPM1L function in maintaining genomic stability?

PPM1L contributes to genomic stability , a critical factor in preventing malignant transformation. Researchers can explore this function using PPM1L antibodies through:

• DNA damage response: Treat cells with DNA-damaging agents and analyze PPM1L expression, phosphorylation state, and localization using immunofluorescence and Western blotting.

• Chromatin association: Perform chromatin immunoprecipitation (ChIP) using PPM1L antibodies to determine if PPM1L associates with chromatin during DNA damage response or replication.

• Interaction with DNA repair proteins: Use co-immunoprecipitation with PPM1L antibodies to identify interactions with components of DNA repair pathways.

• Cell cycle checkpoints: Synchronize cells at different cell cycle phases and analyze PPM1L expression and activity in relation to checkpoint regulators.

• Chromosomal stability assays: Compare chromosomal aberration frequencies between cells with normal and reduced PPM1L levels (using siRNA knockdown verified by PPM1L antibodies).

Understanding PPM1L's role in genomic stability may provide insights into its tumor suppressor potential and involvement in cancer development.

What experimental approaches can reveal novel PPM1L substrates in cellular signaling networks?

Identifying PPM1L substrates is crucial for understanding its role in cellular signaling. Researchers can employ these approaches using PPM1L antibodies:

• Substrate-trapping mutants: Generate phosphatase-dead PPM1L mutants that bind but don't release substrates, immunoprecipitate using PPM1L antibodies, and identify trapped proteins by mass spectrometry.

• Proximity-dependent biotin identification (BioID): Fuse PPM1L to a biotin ligase, express in cells, purify biotinylated proteins, and confirm interactions using PPM1L antibodies in reciprocal co-immunoprecipitation.

• Phosphoproteomic analysis: Compare the phosphoproteome of cells with normal and reduced PPM1L levels (verified by antibodies) to identify hyperphosphorylated proteins that represent potential substrates.

• In vitro dephosphorylation assays: Immunopurify PPM1L using specific antibodies and test its activity against candidate phosphorylated substrates.

• Kinase-phosphatase coupled networks: Analyze the interplay between PPM1L and specific kinases by monitoring phosphorylation dynamics of shared substrates.

• Temporal phosphorylation profiling: After stimulation (e.g., with growth factors or stress inducers), monitor the timing of PPM1L activation relative to substrate dephosphorylation.

These approaches can expand our understanding of PPM1L's role beyond its known interactions with MAP3K7/TAK1 and MAP3K5 .

How can multi-omics approaches integrate PPM1L antibody-based research with other data types?

Integrating PPM1L antibody-based research with multi-omics approaches can provide comprehensive insights into its biological functions:

• Integrative proteomics and phosphoproteomics: Combine PPM1L immunoprecipitation with mass spectrometry to identify both interacting partners and phosphorylation changes dependent on PPM1L activity.

• ChIP-seq correlation: Compare PPM1L chromatin association (if any) determined by ChIP using PPM1L antibodies with transcriptomic changes in response to PPM1L modulation.

• Spatial proteomics: Use immunofluorescence with PPM1L antibodies combined with high-content imaging to map PPM1L's dynamic subcellular distribution in response to various stimuli.

• Protein-metabolite interactions: Investigate whether PPM1L activity (measured using immunoprecipitated PPM1L) is regulated by specific metabolites, connecting phosphatase networks with metabolic states.

• Single-cell correlation analysis: Combine single-cell transcriptomics with immunofluorescence using PPM1L antibodies to correlate protein expression with transcriptional states at the single-cell level.

• Network analysis: Integrate PPM1L interactome data (generated using antibody-based methods) with publicly available protein-protein interaction databases to position PPM1L within larger signaling networks.