ppp4r2b Antibody

What is PPP4R2 and what cellular functions does it regulate?

PPP4R2 (Protein Phosphatase 4 Regulatory Subunit 2) functions as a critical regulatory subunit of serine/threonine-protein phosphatase 4 (PP4). This protein plays several essential roles in cellular homeostasis and stress response mechanisms. PPP4R2 may regulate the activity of PPP4C at centrosomal microtubule organizing centers, suggesting involvement in cell division and cytoskeletal organization . Its interaction with the SMN complex enhances the temporal localization of snRNPs, indicating a role in maturation of spliceosomal snRNPs and RNA processing . Notably, the PPP4C-PPP4R2-PPP4R3A PP4 complex specifically dephosphorylates H2AX phosphorylated on 'Ser-140' (gamma-H2AX) generated during DNA replication and required for DNA double-strand break repair . Additionally, PPP4R2 mediates RPA2 dephosphorylation by recruiting PPP4C to RPA2 in a DNA damage-dependent manner, which is required for the efficient RPA2-mediated recruitment of RAD51 to chromatin following double-strand breaks—an essential step for DNA repair .

What is the difference between PPP4R2 and PPP4R2B antibodies?

While the search results provide comprehensive information about PPP4R2 antibodies, specific information about PPP4R2B antibodies appears limited in the current data. Generally, antibody nomenclature with an appended letter (such as PPP4R2B) often indicates recognition of a specific isoform, splice variant, or post-translationally modified version of the protein.

When selecting between these antibodies, researchers should thoroughly examine the manufacturer's documentation regarding:

• The exact epitope sequence recognized by the antibody

• Whether the antibody targets a specific isoform or all variants

• Cross-reactivity profiles with other protein family members

• Validated experimental applications and species reactivity

For instance, Abcam's PPP4R2 antibody (ab70631) is generated against a synthetic peptide within human PPP4R2 and has been validated for immunoprecipitation (IP), western blotting (WB), and immunohistochemistry on paraffin sections (IHC-P) with human and mouse samples .

What experimental applications are PPP4R2 antibodies validated for?

According to available data, PPP4R2 antibodies have been validated for multiple research applications with varying levels of efficacy. The Abcam PPP4R2 antibody (ab70631) has been specifically tested and validated for:

• Immunoprecipitation (IP): For isolating PPP4R2 and associated protein complexes from cell lysates and tissue homogenates

• Western Blotting (WB): For detecting and quantifying PPP4R2 protein expression in denatured samples

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For visualizing the spatial distribution of PPP4R2 in fixed tissue sections

The antibody demonstrates reactivity with both human and mouse samples, expanding its utility across different model systems . When planning experiments, researchers should consider that for each application, protocol optimization is typically necessary, including determining optimal antibody dilutions, incubation conditions, and appropriate controls to validate specificity.

How should researchers validate PPP4R2 antibody specificity before experimental use?

Rigorous validation of PPP4R2 antibodies is critical for generating reliable research data. A comprehensive validation approach should include:

• Primary Specificity Assessment:

  • Western blot analysis to confirm detection of a single band at the expected molecular weight (~41 kDa)

  • Comparing results from multiple antibodies targeting different epitopes of PPP4R2

  • Testing in samples with genetic manipulation (knockdown/knockout) of PPP4R2 to confirm specificity

• Application-Specific Validation:

  • For Western blotting: Perform peptide competition assays with the immunizing peptide

  • For immunohistochemistry: Compare staining patterns with published data and mRNA expression patterns

  • For immunoprecipitation: Confirm pulled-down protein by mass spectrometry

• Species Cross-Reactivity Testing:

  • Validate in multiple relevant species if cross-species studies are planned

  • The Abcam antibody (ab70631) has been tested on human and mouse samples

• Positive and Negative Controls:

  • Use cell lines or tissues with known high/low expression levels of PPP4R2

  • Include technical controls such as secondary-antibody-only conditions

• Reproducibility Assessment:

  • Repeat validation across multiple batches of the antibody

  • Document lot-to-lot variation for long-term studies

This systematic approach ensures that experimental findings accurately reflect PPP4R2 biology rather than artifacts from non-specific antibody binding.

What are optimal protocol conditions for using PPP4R2 antibodies in immunoprecipitation studies?

Optimizing immunoprecipitation protocols with PPP4R2 antibodies requires careful attention to multiple experimental parameters:

• Lysis Conditions:

  • Use mild, non-denaturing lysis buffers (e.g., RIPA or NP-40-based buffers)

  • Include protease inhibitors to prevent protein degradation

  • Add phosphatase inhibitors to preserve phosphorylation states, particularly important when studying PPP4R2's role in phosphorylation-dependent processes

  • Maintain cold temperature throughout to preserve protein complexes

• Antibody Selection and Amount:

  • Use antibodies validated specifically for immunoprecipitation, such as the Abcam PPP4R2 antibody (ab70631)

  • Optimize antibody amount (typically 2-5 μg per mg of total protein)

  • Pre-incubate antibody with lysate (4-16 hours at 4°C) before adding beads

• Bead Selection and Handling:

  • Choose appropriate beads based on antibody host species (Protein A for rabbit antibodies, Protein G for most mouse antibodies)

  • Pre-clear lysate with beads alone to reduce non-specific binding

  • Consider using magnetic beads for gentler handling and reduced background

• Washing Procedure:

  • Optimize wash buffer stringency—too stringent may disrupt legitimate interactions, too mild may retain non-specific binding

  • Perform 4-5 washes with decreasing salt concentration

  • Consider using detergent gradients in wash buffers

• Controls and Validation:

  • Always include an isotype control antibody IP

  • Verify successful IP by Western blotting for PPP4R2

  • For co-IP studies, confirm known interaction partners like PPP4C or PPP4R3A

This methodology maximizes the chances of successfully isolating physiologically relevant PPP4R2-containing complexes while minimizing artifacts.

How can researchers optimize Western blotting protocols for detecting PPP4R2?

Achieving optimal Western blot results for PPP4R2 detection requires attention to several critical parameters:

• Sample Preparation:

  • Use RIPA or other appropriate lysis buffers with protease and phosphatase inhibitors

  • Ensure complete protein denaturation by heating samples at 95°C for 5 minutes in Laemmli buffer

  • Load appropriate protein amount (typically 20-40 μg of total protein)

• Gel Selection and Electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal resolution of PPP4R2 (~41 kDa)

  • Include positive control samples with known PPP4R2 expression

  • Run at constant voltage (90-120V) to prevent protein degradation

• Transfer Optimization:

  • Use wet transfer systems for more consistent results

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with reversible protein stains like Ponceau S

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

  • Start with manufacturer's recommended antibody dilution (check product datasheet)

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Use appropriate HRP-conjugated secondary antibody specific to host species

• Signal Detection and Troubleshooting:

  • Use enhanced chemiluminescence (ECL) substrate appropriate for expected signal strength

  • For weak signals, consider signal amplification systems or longer exposure times

  • For high background, increase washing steps or further dilute antibodies

Following this methodological approach maximizes sensitivity and specificity when detecting PPP4R2 by Western blotting, allowing for accurate quantification of protein expression levels.

What approaches can be used to investigate PPP4R2's role in DNA damage repair pathways?

Investigating PPP4R2's role in DNA damage repair requires sophisticated experimental approaches that capture both molecular interactions and functional outcomes:

• Microscopy-Based Recruitment Studies:

  • Track fluorescently tagged PPP4R2 recruitment to DNA damage sites using live-cell imaging

  • Quantify colocalization with γ-H2AX and other DNA damage markers

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics at damage sites

  • Apply super-resolution microscopy to resolve the spatial organization of repair complexes

• Molecular Interaction Analysis:

  • Perform time-course co-immunoprecipitation studies following DNA damage induction

  • Use proximity ligation assays to visualize direct interactions between PPP4R2 and repair proteins in situ

  • Apply BioID or APEX2 proximity labeling to identify the damage-dependent interactome

• Functional Assays:

  • Generate PPP4R2-depleted cell lines and rescue with wild-type or mutant constructs

  • Measure γ-H2AX dephosphorylation kinetics using Western blotting and immunofluorescence

  • Assess RPA2 phosphorylation states using phospho-specific antibodies

  • Quantify RAD51 recruitment efficiency in PPP4R2-manipulated cells

• Pathway-Specific Repair Assays:

  • Homologous recombination efficiency using DR-GFP reporter systems

  • Non-homologous end joining using EJ5-GFP reporters

  • Comet assay to measure DNA break repair kinetics

  • Chromosome aberration analysis following DNA damage

• Systems-Level Analysis:

  • Phosphoproteomics to identify PPP4R2-dependent dephosphorylation events following DNA damage

  • ChIP-seq to map PPP4R2 and PPP4C recruitment to chromatin

  • Transcriptomics to assess PPP4R2's impact on DNA damage response gene expression

These approaches collectively provide a comprehensive understanding of how PPP4R2 coordinates the dephosphorylation events necessary for proper DNA damage repair pathway function.

How can researchers investigate PPP4R2's involvement with the SMN complex and spliceosomal machinery?

The role of PPP4R2 in SMN complex function and spliceosomal snRNP maturation represents a complex research area requiring specialized techniques:

• Biochemical Complex Analysis:

  • Perform sequential co-immunoprecipitation to isolate PPP4R2-SMN-containing complexes

  • Use glycerol gradient fractionation to separate distinct SMN-containing complexes

  • Apply analytical size exclusion chromatography to determine complex stoichiometry

  • Conduct in vitro reconstitution assays with purified components

• Temporal Dynamics Assessment:

  • Synchronize cells and analyze PPP4R2-SMN interactions throughout the cell cycle

  • Use fluorescently tagged components and FRAP analysis to measure exchange rates

  • Apply single-molecule tracking to follow individual complexes in living cells

  • Utilize 4D imaging with deconvolution to track snRNP assembly in real time

• Functional Consequences of Disruption:

  • Deplete PPP4R2 using RNAi or CRISPR-Cas9 and measure:

    • snRNP assembly rates using pulse-chase experiments

    • Splicing efficiency using reporter constructs

    • Alternative splicing patterns using RNA-seq

    • Nuclear body formation and dynamics

• Phosphorylation Regulation:

  • Identify phosphorylation sites on SMN complex components using mass spectrometry

  • Generate phosphomimetic and phospho-dead mutants of key residues

  • Assess how phosphorylation status affects complex assembly and function

  • Determine whether PPP4R2's interaction with the SMN complex is phosphorylation-dependent

• Subcellular Localization Studies:

  • Perform immunofluorescence co-localization of PPP4R2 with SMN in nuclear bodies

  • Use Structured Illumination Microscopy (SIM) to resolve sub-nuclear compartments

  • Apply correlative light and electron microscopy to visualize complex ultrastructure

  • Employ biomolecular condensate analysis techniques to study phase separation properties

These methodological approaches provide a comprehensive framework for dissecting PPP4R2's specific roles in the temporal regulation of snRNP localization and maturation.

What methods can be used to study PPP4R2's functions at centrosomal microtubule organizing centers?

Investigating PPP4R2's roles at centrosomal microtubule organizing centers requires specialized techniques that capture both structural and functional aspects:

• High-Resolution Localization Analysis:

  • Perform immunofluorescence with centrosome markers (γ-tubulin, centrin, pericentrin)

  • Apply 3D-Structured Illumination Microscopy (3D-SIM) to resolve PPP4R2's precise localization

  • Use expansion microscopy to physically enlarge centrosomal structures

  • Employ immuno-electron microscopy to determine ultrastructural positioning

• Centrosome Function Assessment:

  • Measure microtubule nucleation capacity using microtubule regrowth assays

  • Analyze centrosome duplication using centrin staining across cell cycle stages

  • Quantify spindle formation and structure in PPP4R2-depleted cells

  • Assess chromosome segregation errors using live-cell imaging

• PPP4R2 Recruitment Dynamics:

  • Generate cell lines expressing fluorescently tagged PPP4R2

  • Perform live-cell imaging throughout the cell cycle

  • Measure protein turnover at centrosomes using photobleaching techniques

  • Apply optogenetic approaches to acutely recruit or remove PPP4R2 from centrosomes

• Substrate Identification and Validation:

  • Perform BioID proximity labeling with PPP4R2 as bait at centrosomes

  • Use quantitative phosphoproteomics to identify PPP4R2-dependent substrates

  • Validate candidates using phospho-specific antibodies

  • Generate and test phosphomimetic/phospho-dead mutants of key substrates

• Functional Disruption Approaches:

  • Generate PPP4R2 knockout/knockdown cell lines

  • Create centrosome-targeted PPP4R2 using PACT domain fusion

  • Express dominant-negative PPP4R2 mutants

  • Rescue experiments with wild-type vs. mutant PPP4R2 constructs

These approaches collectively enable a comprehensive characterization of PPP4R2's role in regulating centrosomal protein phosphorylation status and subsequent functional outcomes .

How should researchers interpret contradictory results from different PPP4R2 antibodies?

When researchers encounter contradictory results using different PPP4R2 antibodies, a systematic analytical approach is essential:

• Epitope Analysis and Accessibility Considerations:

  • Map the epitopes recognized by each antibody (e.g., the Abcam antibody ab70631 targets a specific synthetic peptide within human PPP4R2)

  • Consider whether epitopes may be masked by:

    • Protein-protein interactions in multiprotein complexes

    • Post-translational modifications near the epitope

    • Conformational changes under different experimental conditions

    • Fixation-induced epitope masking (particularly relevant for IHC/ICC)

• Antibody Validation Assessment:

  • Review validation data for each antibody, including Western blot profiles

  • Check for validation in knockout/knockdown systems

  • Evaluate published literature using each antibody

  • Consider performing peptide competition assays to confirm specificity

• Application-Specific Performance:

  • Categorize results by application (WB, IP, IHC) as antibodies may perform differently across techniques

  • Assess whether contradictions appear only in specific applications

  • Optimize protocols for each antibody independently

  • Consider using polyclonal and monoclonal antibodies in parallel

• Biological Variable Consideration:

  • Evaluate whether contradictions might reflect actual biological differences:

    • Detection of different isoforms

    • Cell-type specific post-translational modifications

    • Presence/absence of interacting proteins affecting epitope accessibility

• Resolution Strategy:

  • Generate a consensus result using multiple antibodies targeting different epitopes

  • Implement orthogonal detection methods not relying on antibodies

  • Consider genetic tagging approaches (HA, FLAG, GFP) for unambiguous detection

  • Document and report all contradictions transparently in publications

What are common technical challenges in detecting PPP4R2 by immunohistochemistry?

Immunohistochemical detection of PPP4R2 presents several technical challenges that researchers should anticipate and address:

• Tissue Fixation and Processing Issues:

  • Overfixation can mask epitopes through excessive protein cross-linking

  • Underfixation can lead to poor tissue morphology and antigen loss

  • Optimize fixation protocols with different fixatives (4% PFA, Bouin's solution)

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

• Antibody Selection and Validation:

  • Choose antibodies specifically validated for IHC-P, such as the Abcam PPP4R2 antibody (ab70631)

  • Evaluate lot-to-lot variability that may affect staining consistency

  • Include positive control tissues with known PPP4R2 expression

  • Use PPP4R2-depleted tissues as negative controls when possible

• Signal Amplification and Detection Challenges:

  • Low-abundance proteins may require signal amplification systems

  • Test polymer-based detection systems for improved sensitivity

  • Optimize chromogen development time to balance signal and background

  • Consider tyramide signal amplification for very low abundance targets

• Background Reduction Strategies:

  • Implement stringent blocking procedures (3-5% BSA, normal serum matching secondary antibody species)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific hydrophobic interactions

  • Use avidin/biotin blocking for tissues with endogenous biotin

  • Employ hydrogen peroxide pre-treatment to quench endogenous peroxidases

• Multiplexing Considerations:

  • When co-staining with other markers, test antibody compatibility

  • Optimize sequential staining protocols if cross-reactivity occurs

  • Consider spectral unmixing approaches for overlapping signals

  • Use appropriate controls for multiplex staining

Addressing these technical challenges systematically improves the reliability and reproducibility of PPP4R2 detection in tissue sections, enabling more accurate assessment of expression patterns across different tissues and disease states.

How should researchers approach troubleshooting non-specific bands in PPP4R2 Western blots?

Non-specific bands in PPP4R2 Western blots can significantly complicate data interpretation. A methodical troubleshooting approach includes:

• Sample Preparation Optimization:

  • Test different lysis buffers (RIPA, NP-40, Triton X-100) to minimize protein degradation

  • Add fresh protease inhibitors immediately before lysis

  • Maintain samples at 4°C throughout preparation

  • Centrifuge lysates at high speed to remove cell debris

• Electrophoresis and Transfer Conditions:

  • Optimize gel percentage (10-12% is typically appropriate for PPP4R2)

  • Ensure complete protein denaturation (95°C for 5 minutes in reducing buffer)

  • Calibrate transfer conditions (voltage, time, buffer composition)

  • Verify transfer efficiency using reversible staining

• Antibody-Specific Strategies:

  • Titrate primary antibody to determine optimal concentration

  • Extend washing steps (5-6 washes, 10 minutes each)

  • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

  • Consider switching to a more specific antibody or different clone

• Validation Experiments:

  • Run PPP4R2-depleted samples (siRNA or CRISPR knockout) to identify the specific band

  • Perform peptide competition assays with the immunizing peptide

  • Compare patterns across multiple antibodies targeting different PPP4R2 epitopes

  • Consider phosphatase treatment if phosphorylation creates multiple bands

• Interpretation Framework:

  • Create a molecular weight map of expected PPP4R2 isoforms and fragments

  • Document consistent non-specific bands across experiments

  • Consider whether apparent non-specific bands might represent:

    • PPP4R2 isoforms or splice variants

    • Post-translationally modified forms

    • Degradation products

    • Cross-reactivity with related protein family members

This systematic approach helps distinguish true PPP4R2 signal from artifacts, ensuring reliable protein detection and quantification in Western blotting applications.

How can researchers design experiments to study PPP4R2's role in RPA2-mediated recruitment of RAD51?

Investigating the role of PPP4R2 in RPA2-mediated recruitment of RAD51 requires a multifaceted experimental approach:

• Genetic Manipulation Strategies:

  • Generate PPP4R2 knockout/knockdown cell lines using CRISPR-Cas9 or RNAi

  • Create complementation systems expressing wild-type or phosphatase-dead PPP4R2 mutants

  • Develop inducible depletion systems for temporal control of PPP4R2 levels

  • Engineer cell lines expressing fluorescently tagged RPA2 and RAD51

• DNA Damage Induction Methods:

  • Use ionizing radiation for random DSB induction

  • Apply laser microirradiation for spatially defined damage

  • Utilize site-specific endonucleases for targeted genomic breaks

  • Employ replication stress inducers (hydroxyurea, aphidicolin) to study replication-associated damage

• Protein Interaction and Localization Analysis:

  • Perform co-immunoprecipitation of PPP4R2, PPP4C, RPA2, and RAD51 before and after damage

  • Use proximity ligation assays to visualize protein interactions in situ

  • Employ ChIP-seq to map protein recruitment to chromatin

  • Apply super-resolution microscopy to visualize spatial organization at damage sites

• Phosphorylation Analysis:

  • Utilize phospho-specific antibodies to monitor RPA2 phosphorylation status

  • Perform in vitro dephosphorylation assays with purified components

  • Generate phosphomimetic and phospho-dead RPA2 mutants

  • Apply quantitative mass spectrometry to identify all phosphorylation sites

• Functional Outcome Assessment:

  • Measure RAD51 foci formation in PPP4R2-depleted vs. control cells

  • Quantify homologous recombination efficiency using reporter assays

  • Assess DNA repair kinetics using comet assays

  • Determine cell survival following DNA damage

This comprehensive experimental framework allows researchers to dissect the specific role of PPP4R2 in mediating RPA2 dephosphorylation and subsequent RAD51 recruitment to chromatin following double-strand breaks, an essential step for DNA repair .

What experimental approaches can be used to study the role of PPP4R2 in H2AX dephosphorylation?

Investigating PPP4R2's role in H2AX dephosphorylation requires sophisticated experimental approaches spanning multiple techniques:

• Phosphorylation Dynamics Analysis:

  • Perform time-course Western blotting with phospho-specific γ-H2AX antibodies

  • Use quantitative immunofluorescence to measure γ-H2AX foci resolution kinetics

  • Apply flow cytometry to quantify γ-H2AX levels at population scale

  • Employ ELISA-based methods for precise phosphorylation quantification

• Complex Component Manipulation:

  • Selectively deplete PPP4R2, PPP4C, or PPP4R3A to dissect complex dependencies

  • Create separation-of-function mutants disrupting specific protein interactions

  • Express dominant-negative PPP4R2 variants to inhibit endogenous activity

  • Use degron systems for rapid, inducible protein depletion

• Direct Biochemical Interaction Studies:

  • Perform in vitro dephosphorylation assays with reconstituted components

  • Use surface plasmon resonance to measure binding affinities between components

  • Apply hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Conduct co-crystallization studies of PPP4R2 with substrate peptides

• Chromatin Association Analysis:

  • Use ChIP-seq to map PPP4R2 recruitment to γ-H2AX-enriched chromatin regions

  • Apply CUT&RUN for high-resolution protein localization

  • Employ sequential ChIP to identify regions with co-occurrence of multiple factors

  • Analyze chromatin states associated with PPP4R2 recruitment

• Experimental Design Table:

This comprehensive experimental framework enables detailed characterization of how the PPP4C-PPP4R2-PPP4R3A complex specifically dephosphorylates H2AX phosphorylated on 'Ser-140' (gamma-H2AX) during DNA replication and repair processes .

How can researchers investigate the centrosomal functions of PPP4R2 using advanced microscopy techniques?

Advanced microscopy approaches offer powerful tools for investigating PPP4R2's functions at centrosomal microtubule organizing centers:

• Super-Resolution Microscopy Applications:

  • Employ 3D-Structured Illumination Microscopy (3D-SIM) to resolve PPP4R2 localization within centrosomal substructures

  • Apply STORM/PALM for single-molecule localization at nanometer resolution

  • Use expansion microscopy to physically enlarge centrosomes for improved resolution

  • Implement live-cell super-resolution to track dynamic processes

• Live-Cell Imaging Strategies:

  • Generate cell lines expressing fluorescently tagged PPP4R2 (e.g., PPP4R2-GFP)

  • Perform time-lapse microscopy throughout the cell cycle

  • Implement photobleaching techniques (FRAP, FLIP) to measure protein dynamics

  • Use fluorescent speckle microscopy to track microtubule growth from centrosomes

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence microscopy of PPP4R2 with electron microscopy

  • Use immunogold labeling for ultrastructural localization

  • Apply electron tomography for 3D reconstruction of centrosome architecture

  • Integrate with focused ion beam scanning electron microscopy for volume imaging

• Quantitative Image Analysis Approaches:

  • Develop custom algorithms for centrosome feature extraction

  • Apply machine learning for automated detection of centrosomal abnormalities

  • Implement 3D object-based colocalization analysis

  • Use particle tracking to measure microtubule nucleation rates

• Functional Perturbation Imaging:

  • Combine optogenetic protein recruitment/removal with live imaging

  • Apply laser ablation to disrupt centrosome integrity and monitor recovery

  • Implement local photoactivation of caged compounds to modulate PPP4R2 activity

  • Use fluorescent biosensors to monitor phosphorylation status in real-time

These advanced microscopy approaches provide unprecedented insights into how PPP4R2 may regulate the activity of PPP4C at centrosomal microtubule organizing centers , revealing both structural organization and dynamic functional properties at high spatiotemporal resolution.