PPP5C Monoclonal Antibody

What is PPP5C and what cellular functions does it regulate?

PPP5C (Protein Phosphatase 5 Catalytic subunit) is a serine/threonine phosphatase belonging to the protein phosphatase catalytic subunit family. It functions primarily in pathways regulated by reversible phosphorylation at serine and threonine residues, many of which control cell growth and differentiation processes . The protein participates in signaling pathways responding to hormones and cellular stress conditions .

PPP5C is known to regulate several important biological processes:

• Signal transduction through the MAPK pathway

• Stress response signaling

• Cell growth and differentiation regulation

• Calcium channel activity modulation through interaction with ISOC (store-operated calcium channels)

• Negative regulation of glucocorticoid receptor signaling

Additionally, dysregulation of PPP5C has been implicated in disease states, with elevated levels potentially associated with breast cancer development .

What are the key specifications of commercially available PPP5C monoclonal antibodies?

PPP5C monoclonal antibodies are available in different clones with varying specifications for research applications. The following table summarizes key characteristics of two prominent clones:

Both antibodies recognize the full-length PPP5C protein and are purified using affinity chromatography (protein A/G) from either mouse ascites fluids or tissue culture supernatant .

How should PPP5C antibodies be stored and handled to maintain optimal activity?

To maintain optimal activity of PPP5C monoclonal antibodies, researchers should follow these evidence-based storage and handling protocols:

• Temperature: Store antibodies at -20°C as received in the manufacturer's packaging .

• Buffer composition: PPP5C antibodies are typically supplied in PBS (pH 7.3) containing 1% BSA, 50% glycerol, and 0.02% sodium azide . This formulation helps maintain stability during freeze-thaw cycles.

• Stability period: When stored properly, the antibodies are stable for 12 months from the date of receipt .

• Shipping conditions: The antibodies are shipped on blue ice to maintain integrity during transport .

• Aliquoting: For frequent users, it's recommended to prepare small aliquots upon receipt to minimize freeze-thaw cycles.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment according to the recommended dilution ratios for specific applications (WB, IHC, FC, or IF) .

• Safety considerations: Note that the buffer contains sodium azide, which is toxic. Handle with appropriate precautions and dispose of according to safety regulations.

What are the optimized protocols for different applications of PPP5C monoclonal antibodies?

Different experimental applications require specific protocols for optimal results with PPP5C monoclonal antibodies:

Western Blot (WB) Protocol:

• Sample preparation: Prepare cell or tissue lysates using standard RIPA buffer with protease inhibitors.

• Protein separation: Load 20-50 μg of protein per lane on 10-12% SDS-PAGE gels.

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes.

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

• Primary antibody: Dilute PPP5C antibody at 1:250-1000 (depending on clone) in blocking buffer and incubate overnight at 4°C.

• Washing: Wash 3× with TBST, 5 minutes each.

• Secondary antibody: Incubate with HRP-conjugated anti-mouse IgG at 1:5000 for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence substrate.

• Expected result: A single band at approximately 56.7 kDa .

Immunohistochemistry (IHC) Protocol:

• Sample preparation: Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thick).

• Deparaffinization and rehydration: Standard xylene and ethanol series.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0).

• Blocking: 3% hydrogen peroxide followed by protein block.

• Primary antibody: Dilute PPP5C antibody at 1:50 and incubate overnight at 4°C.

• Detection: Use a polymer detection system appropriate for mouse primary antibodies.

• Counterstain: Hematoxylin.

• Expected result: Variable staining patterns depending on tissue type and PPP5C expression levels.

Immunofluorescence (IF) Protocol:

• Sample preparation: Fix cells with 4% paraformaldehyde for 15 minutes.

• Permeabilization: 0.1% Triton X-100 for 10 minutes.

• Blocking: 5% normal serum in PBS for 30 minutes.

• Primary antibody: Dilute PPP5C antibody at 1:100 and incubate overnight at 4°C.

• Secondary antibody: Fluorophore-conjugated anti-mouse IgG at 1:500 for 1 hour at room temperature.

• Counterstain: DAPI for nuclear visualization.

• Mounting: Anti-fade mounting medium.

How can I validate the specificity of PPP5C antibodies in my experimental system?

Validating antibody specificity is critical for ensuring reliable research results. For PPP5C monoclonal antibodies, implement these validation strategies:

• Genetic manipulation controls:

  • Use PP5C-/- cell lines as negative controls, as described in research where PP5C expression was disrupted using CRISPR-Cas9 .

  • Compare with wild-type and PP5C-reintroduced cells to confirm specificity .

• Knockdown validation:

  • Perform siRNA-mediated suppression of PP5C expression .

  • Compare antibody signal between knockdown and control samples.

• Peptide competition assay:

  • Pre-incubate the antibody with excess purified recombinant PPP5C protein.

  • A specific antibody will show reduced or eliminated signal in subsequent applications.

• Multiple antibody validation:

  • Compare results using different PPP5C antibody clones (e.g., OTI6C2 and OTI2G2) .

  • Concordant results strengthen confidence in specificity.

• Signal correlation with known expression patterns:

  • Verify that detected signal corresponds with tissues/cells known to express PPP5C.

  • Check subcellular localization against known distribution patterns.

• Molecular weight verification:

  • Confirm that the detected band in Western blot corresponds to the predicted 56.7 kDa size of PPP5C .

  • Check for potential post-translational modifications that might alter apparent molecular weight.

• Cross-reactivity testing:

  • Test antibody against closely related phosphatases to ensure specificity.

What controls should be included when working with PPP5C antibodies?

When designing experiments using PPP5C monoclonal antibodies, include these essential controls:

• Positive controls:

  • Cell lines or tissues with confirmed PPP5C expression

  • Recombinant PPP5C protein (such as the full-length human recombinant protein used as immunogen)

• Negative controls:

  • PP5C-/- cell lines generated through CRISPR-Cas9 gene editing

  • Primary antibody omission control

  • Isotype control (matching IgG1 or IgG2b, depending on the clone used)

• Loading/technical controls:

  • Housekeeping protein detection (e.g., β-actin, GAPDH) for Western blots

  • Tissue-specific markers for IHC/IF to verify tissue integrity

• Functional controls:

  • Paired wild-type and catalytically inactive PP5C (PP5C-CatΔ) for functional studies

  • Phosphorylation state-specific controls when studying PP5C substrates

• Dose-dependent controls:

  • Serial dilution of primary antibody to determine optimal concentration

  • Titration experiments to verify signal specificity

• Cross-species validation:

  • When working with non-human samples, verify antibody reactivity in that species (antibodies show reactivity with dog, human, monkey, mouse, rat)

• Subcellular fractionation controls:

  • In studies examining PP5C subcellular localization, include markers for different cellular compartments

How does the structural basis of PPP5C inform its catalytic mechanism?

The high-resolution crystal structure of PPP5C at 1.6 Å provides critical insights into its catalytic mechanism and exceptional enzymatic efficiency:

The PPP5C catalytic domain contains a distinctive Asp271-M1:M2-W1-His427-His304-Asp274 motif that is essential for its phosphatase activity . This arrangement creates a unique metal-binding pocket that orchestrates the hydrolysis of phosphoprotein substrates through:

• Bimetal mechanism: Two metal ions (M1 and M2, typically Mn2+ or Fe2+) are precisely positioned within the catalytic pocket . These metals:

  • Activate a water molecule (W1) for nucleophilic attack

  • Stabilize the transition state during phosphate hydrolysis

  • Coordinate with conserved aspartate and histidine residues

• Nucleophilic activation: The catalytic water molecule (W1) positioned between the two metal ions becomes highly nucleophilic, enabling attack on the phosphorus atom of the substrate .

• Substrate binding pocket: The structure reveals a surface groove that accommodates phosphoprotein substrates, with specificity determined by:

  • The depth and width of the binding pocket

  • Charge distribution within the catalytic site

  • Interactions with conserved residues in the active site

• C-terminal subdomain: The structural analysis revealed a previously unidentified subdomain in the C-terminus that likely contributes to:

  • Substrate recognition

  • Protein-protein interactions

  • Regulatory functions

Understanding this structural basis helps explain why PPP5C and related phosphatases are among the most catalytically efficient enzymes known, with implications for designing specific inhibitors for research and potential therapeutic applications .

What is the role of PPP5C in calcium channel regulation and endothelial barrier function?

PPP5C plays a crucial role in calcium channel regulation and endothelial barrier function through several molecular mechanisms:

• ISOC channel regulation:

  • PPP5C is an essential component of the store-operated calcium channel (ISOC) heterocomplex .

  • It co-localizes with TRPC4, an essential subunit of ISOC channels, as demonstrated by co-precipitation studies .

  • PPP5C is found in the same membrane fractions as the ISOC heterocomplex in pulmonary artery endothelial cells (PAECs) .

• FKBP51-mediated inhibition:

  • PPP5C is required for FKBP51-mediated inhibition of ISOC channels .

  • siRNA-mediated suppression of PP5C expression attenuates FKBP51's ability to inhibit ISOC activity .

  • Genetic disruption of PP5C in HEK293 cells similarly prevents FKBP51-mediated ISOC inhibition .

• Catalytic activity requirement:

  • The phosphatase activity of PPP5C is essential for its regulatory function:

  • Reintroduction of catalytically competent PP5C restores FKBP51-mediated inhibition of ISOC .

  • Catalytically inactive PP5C fails to restore this function .

• Endothelial barrier protection:

  • PPP5C contributes to endothelial barrier integrity by preventing calcium entry-induced inter-endothelial cell gap formation .

  • Suppression of PP5C expression negates the protective effect of FKBP51 on endothelial barrier function .

  • This protective effect was confirmed through multiple methodologies:

    • Wide-field microscopy for gap area measurement

    • Biotin gap quantification assay

    • Electric cell-substrate impedance sensing (ECIS®)

• Potential mechanisms:

  • PPP5C likely regulates ISOC activity through dephosphorylation of key channel components or regulatory proteins.

  • Similar to its role in neurons where it promotes microtubule stability through tau dephosphorylation, PPP5C may influence cytoskeletal dynamics in endothelial cells .

This research has significant implications for understanding vascular permeability regulation and potential therapeutic targets for conditions involving endothelial barrier dysfunction, such as acute respiratory distress syndrome or pulmonary edema.

How can PPP5C antibodies be used to investigate its role in cellular stress response pathways?

PPP5C antibodies can be employed in sophisticated experimental approaches to elucidate its role in cellular stress response pathways:

• Stress-induced phosphorylation changes:

  • Use PPP5C antibodies in conjunction with phospho-specific antibodies to analyze how various stressors (oxidative stress, heat shock, DNA damage) affect PPP5C substrates.

  • Implement quantitative Western blot analysis to track temporal changes in PPP5C expression and localization following stress induction.

  • Compare these changes between wild-type cells and those expressing catalytically inactive PPP5C mutants .

• Protein-protein interaction networks:

  • Perform co-immunoprecipitation with PPP5C antibodies followed by mass spectrometry to identify stress-specific interaction partners.

  • Use proximity ligation assays (PLA) to visualize and quantify in situ interactions between PPP5C and stress response proteins like heat shock proteins.

  • Investigate the dynamic assembly of PPP5C-containing complexes through size-exclusion chromatography followed by Western blotting.

• Subcellular trafficking:

  • Track PPP5C redistribution during stress responses using immunofluorescence with PPP5C antibodies .

  • Implement live-cell imaging with fluorescently-tagged PPP5C in combination with immunostaining of fixed time points.

  • Use subcellular fractionation followed by Western blotting to quantify PPP5C redistribution between cellular compartments during stress.

• MAPK pathway regulation:

  • Since PPP5C participates in MAPK signaling , use PPP5C antibodies to investigate its association with pathway components during stress.

  • Perform phosphatase activity assays on immunoprecipitated PPP5C to measure stress-induced changes in enzymatic activity.

  • Compare phosphorylation states of MAPK pathway components between normal and PPP5C-depleted cells under stress conditions.

• Hormonal stress responses:

  • Investigate PPP5C's role in glucocorticoid receptor (GR) signaling using hormone stimulation experiments.

  • Monitor PPP5C-GR interactions using co-immunoprecipitation with PPP5C antibodies.

  • Analyze how stress-induced hormonal changes affect PPP5C expression, localization, and activity.

• ChIP-seq applications:

  • Use chromatin immunoprecipitation (ChIP) with PPP5C antibodies to identify potential chromatin-associated functions during stress responses.

  • Investigate whether PPP5C directly or indirectly regulates stress-responsive gene expression.

What are common technical issues with PPP5C antibodies and how can they be resolved?

Researchers may encounter several technical challenges when working with PPP5C monoclonal antibodies. Here are common issues and evidence-based solutions:

• High background in immunostaining:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution:

    • Increase blocking time and concentration (try 5-10% serum or BSA)

    • Further dilute primary antibody (start with manufacturer's recommended 1:50 for IHC )

    • Extend washing steps (5 × 5 minutes with gentle agitation)

    • Add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific membrane binding

• Multiple bands in Western blot:

  • Cause: Cross-reactivity, degradation products, splice variants, or post-translational modifications

  • Solution:

    • Verify sample preparation (add fresh protease inhibitors)

    • Compare with PP5C-/- control samples

    • Use gradient gels to better resolve protein species

    • Consider using reducing vs. non-reducing conditions

    • Remember that alternative splicing of PPP5C results in multiple transcript variants

• Weak or no signal:

  • Cause: Low expression levels, epitope masking, over-fixation, or antibody degradation

  • Solution:

    • Optimize antigen retrieval for IHC (try citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

    • Increase antibody concentration or incubation time

    • Try different lysis buffers for Western blot

    • Test antibody on positive control samples with known PPP5C expression

    • Verify antibody stability and storage conditions

• Inconsistent results between experiments:

  • Cause: Antibody batch variation, protocol inconsistency, or sample handling

  • Solution:

    • Standardize protocols with precise timing and temperature controls

    • Prepare larger antibody working dilution aliquots to use across experiments

    • Include consistent positive controls in each experiment

    • Consider using two different PPP5C antibody clones (OTI6C2 and OTI2G2)

• Poor immunoprecipitation efficiency:

  • Cause: Insufficient antibody-antigen binding, buffer incompatibility

  • Solution:

    • Pre-clear lysates to reduce non-specific binding

    • Optimize antibody-to-bead and antibody-to-lysate ratios

    • Extend binding time (overnight at 4°C)

    • Try different detergent concentrations in IP buffer

How can I distinguish between PPP5C and other similar phosphatases in my experiments?

Distinguishing PPP5C from other similar phosphatases requires careful experimental design and multiple validation approaches:

• Structural and sequence-based discrimination:

  • PPP5C has distinctive tetratricopeptide repeat (TPR) domains in its N-terminus that differentiate it from other phosphatases .

  • Its molecular weight (56.7 kDa) differs from other phosphatases, aiding identification in Western blots.

  • PPP5C's unique C-terminal subdomain revealed by crystallography can be targeted with domain-specific antibodies.

• Antibody-based discrimination:

  • Use antibodies generated against unique epitopes not present in other phosphatases.

  • Validate antibody specificity through immunoprecipitation followed by mass spectrometry.

  • Perform cross-reactivity tests with recombinant proteins of related phosphatases.

• Functional discrimination:

  • Utilize specific PP5C inhibitors like okadaic acid (which has differential IC50 values for different phosphatases).

  • Develop activity assays using PPP5C-specific substrates or substrate peptides.

  • Compare phosphatase activity in wild-type versus PP5C-/- samples .

• Localization-based discrimination:

  • Unlike some other phosphatases, PPP5C shows specific subcellular distribution patterns.

  • Use co-localization studies with organelle markers to distinguish PPP5C from other phosphatases.

  • Examine co-localization with known PPP5C interactors like TRPC4 .

• Molecular genetic approaches:

  • Generate cell lines with epitope-tagged versions of PPP5C for unambiguous identification.

  • Use CRISPR-Cas9 gene editing to create PP5C knockout lines as negative controls .

  • Employ isoform-specific siRNA to selectively knockdown PPP5C.

• Biochemical separation:

  • Use ion exchange chromatography to separate phosphatases based on charge differences.

  • Implement size exclusion chromatography to distinguish PPP5C-containing complexes.

  • Design immunodepletion experiments to selectively remove PPP5C from samples.

• Association with specific binding partners:

  • PPP5C's TPR domains mediate specific interaction with heat shock protein-90 (HSP90) .

  • Investigate co-immunoprecipitation with HSP90 or FKBP51 as a distinguishing feature.

What quantitative methods are most appropriate for analyzing PPP5C expression and activity?

Several quantitative methods can be employed to analyze PPP5C expression and activity with high precision:

• Expression quantification methods:

  Western blot densitometry:

  • Suitable for semi-quantitative analysis of PPP5C protein levels.

  • Normalize to appropriate loading controls (β-actin, GAPDH).

  • Use digital imaging systems with linear dynamic range.

  • Implement standard curves with recombinant PPP5C protein for absolute quantification.

  qRT-PCR:

  • Quantify PPP5C mRNA expression levels.

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification.

  • Use the 2^(-ΔΔCT) method with appropriate reference genes.

  • Consider potential alternative splice variants of PPP5C .

  ELISA:

  • Develop sandwich ELISA using two different epitope-targeting antibodies.

  • Establish standard curves with recombinant PPP5C protein.

  • Offers higher throughput than Western blotting.

  Mass spectrometry:

  • For absolute quantification, implement multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM).

  • Use isotopically labeled peptide standards (AQUA peptides) specific to PPP5C.

  • Enables simultaneous quantification of post-translational modifications.

• Activity quantification methods:

  Phosphatase activity assays:

  • Immunoprecipitate PPP5C using specific antibodies .

  • Measure activity using artificial substrates like p-nitrophenyl phosphate (pNPP).

  • For more specificity, use phosphopeptides based on known PPP5C substrates.

  • Include controls with catalytically inactive PPP5C mutants .

  Cellular reporter systems:

  • Develop FRET-based biosensors for real-time monitoring of PPP5C activity.

  • Use phosphorylation-responsive luciferase reporters linked to PPP5C substrates.

  • Implement these systems in wild-type versus PP5C-/- backgrounds .

  Phospho-specific Western blotting:

  • Monitor phosphorylation states of known PPP5C substrates.

  • Quantify changes in phosphorylation upon PPP5C manipulation.

  • Include time-course analyses to capture dynamics.

  Phosphoproteomics:

  • Compare global phosphorylation changes between normal and PPP5C-deficient cells.

  • Implement stable isotope labeling (SILAC or TMT) for quantitative comparison.

  • Focus on serine/threonine phosphorylation sites affected by PPP5C activity.

• Localization and interaction quantification:

  Quantitative immunofluorescence:

  • Measure PPP5C intensity in different subcellular compartments.

  • Use automated image analysis software for unbiased quantification.

  • Implement co-localization coefficients to quantify association with other proteins.

  Proximity ligation assay (PLA):

  • Quantify in situ PPP5C interactions with proteins like TRPC4 or HSP90.

  • Provides spatial information about protein interactions.

  • Allows quantification of interaction events per cell.

  FRAP (Fluorescence Recovery After Photobleaching):

  • Measure PPP5C mobility and binding dynamics in living cells.

  • Quantify changes in mobility upon cellular stress or stimulation.

How might PPP5C be implicated in disease mechanisms and potential therapeutic approaches?

PPP5C's involvement in multiple signaling pathways positions it as a potentially significant factor in various disease mechanisms and therapeutic strategies:

What innovative methodologies are being developed for studying PPP5C phosphatase activity?

Cutting-edge methodologies are expanding our ability to study PPP5C phosphatase activity with unprecedented precision:

• CRISPR-based approaches:

  • CRISPR-Cas9 gene editing has been used to generate PP5C-/- cell lines for functional studies .

  • Advanced applications include:

    • CRISPR activation (CRISPRa) and interference (CRISPRi) for temporal control of PPP5C expression

    • Base editing for introducing specific catalytic mutations without disrupting expression

    • Prime editing for precise modification of regulatory sequences

• Optogenetic control systems:

  • Light-inducible PPP5C activation/inactivation systems allow:

    • Spatiotemporal control of phosphatase activity

    • Reversible modulation of activity in specific subcellular compartments

    • Real-time correlation between activity changes and cellular responses

• Single-molecule approaches:

  • Super-resolution microscopy techniques (STORM, PALM) to visualize individual PPP5C molecules

  • Single-molecule FRET to monitor conformational changes during substrate binding

  • Single-cell phosphoproteomics to analyze cell-to-cell variability in PPP5C activity

• Microfluidic platforms:

  • Droplet-based single-cell analysis of PPP5C activity

  • Gradient generators to study PPP5C response to varying concentrations of stimuli

  • Organ-on-chip models for tissue-specific PPP5C function analysis

• Biosensor development:

  • Genetically encoded FRET-based sensors specific for PPP5C activity

  • Split-luciferase complementation systems for monitoring PPP5C-substrate interactions

  • Chemiluminescent probes for non-invasive imaging of PPP5C activity in vivo

• Structural biology advances:

  • Cryo-EM analysis of PPP5C in complex with regulatory proteins and substrates

  • Building upon the existing 1.6 Å crystal structure to design:

    • Structure-based inhibitors with enhanced specificity

    • Modified substrates for mechanistic studies

    • Engineered PPP5C variants with altered specificity

• Systems biology approaches:

  • Network analysis of PPP5C interactome under different cellular conditions

  • Integration of phosphoproteomics, transcriptomics, and metabolomics data

  • Computational modeling of PPP5C-regulated signaling networks

• Protein engineering methods:

  • Chemical genetic approaches with engineered PPP5C variants sensitive to specific inhibitors

  • Catalytically inactive PPP5C variants that act as substrate traps

  • Split-PPP5C complementation systems for studying protein-protein interactions