PPP4R2 Antibody

What is PPP4R2 and why is it a significant research target?

PPP4R2 (Protein Phosphatase 4 Regulatory Subunit 2) functions as a regulatory subunit of protein phosphatase-4 (PPP4), a serine/threonine phosphatase with critical roles in cellular processes. PPP4R2 governs the dephosphorylation activity and substrate specificity of the PPP4 complex . It is particularly significant in research due to its involvement in:

• DNA damage response (DDR) and repair mechanisms

• Microtubule organization at centrosomes

• Cell development and differentiation

• Apoptotic pathways

• Tumor progression and suppression mechanisms

Research interest in PPP4R2 has intensified following the discovery of its recurrent deletion in acute myeloid leukemia (AML), suggesting a potential tumor suppressor role .

What types of PPP4R2 antibodies are currently available for research applications?

Current research-grade PPP4R2 antibodies include:

• Polyclonal antibodies:

  • Rabbit-derived polyclonal antibodies (e.g., 22676-1-AP from Proteintech, SAB4500565 from Sigma-Aldrich, A10882-1 from Boster)

  • These target multiple epitopes and are useful for various applications

• Antibodies categorized by application compatibility:

  Application	Available Antibodies	Typical Dilution Ranges
  Western Blot (WB)	Proteintech 22676-1-AP, Sigma SAB4500565	1:500-1:2000
  Immunohistochemistry (IHC)	Proteintech 22676-1-AP	1:20-1:200
  Immunofluorescence (IF)/ICC	Proteintech 22676-1-AP	1:50-1:500
  Immunoprecipitation (IP)	Proteintech 22676-1-AP	0.5-4.0 μg for 1.0-3.0 mg lysate
  Co-IP	Proteintech 22676-1-AP	Varies by protocol
  ELISA	Sigma SAB4500565, Boster A10882-1	1:5000

• Species reactivity:

  • Human-reactive: Proteintech 22676-1-AP, Sigma SAB4500565, Boster A10882-1

  • Non-human primate-reactive: Boster A10882-1

How can I validate the specificity of a PPP4R2 antibody for my research?

A robust validation strategy for PPP4R2 antibodies should include:

• Western blot analysis:

  • Expected molecular weight: The observed molecular weight for PPP4R2 is approximately 65 kDa, though calculated weights of 40-55 kDa have been reported

  • Positive controls: A549 cells and HepG2 cells have been validated for WB applications

  • Include negative controls such as PPP4R2-knockdown cell lines when possible

• Immunohistochemistry validation:

  • Positive tissue controls: Human liver cancer tissue and human pancreas tissue have been validated

  • Antigen retrieval optimization: Test both TE buffer pH 9.0 and citrate buffer pH 6.0

• RNAi-based validation:

  • Use PPP4R2-knockdown samples as specificity controls

  • Look for decreased signal corresponding to protein reduction

• Cell line panel testing:

  • Test antibody performance across multiple cell lines with known PPP4R2 expression levels

  • Correlate protein detection with mRNA expression data when available

• Phosphoprotein detection:

  • For studies of DDR mechanisms, validate with IR-treated samples to detect changes in phosphorylated substrate proteins (pKAP1, γH2AX)

What are the optimal conditions for using PPP4R2 antibodies in western blotting experiments?

For optimal western blotting with PPP4R2 antibodies:

• Sample preparation:

  • Recommended positive control cell lines: A549 and HepG2 cells

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • For phosphorylation studies, include phosphatase inhibitors in your lysis buffer

• Protein loading and separation:

  • Load 20-40 μg of total protein per lane

  • Use 10% SDS-PAGE gels for optimal separation around 65 kDa (observed MW)

• Transfer and blocking:

  • PVDF membranes are recommended for optimal protein binding

  • Block with 5% non-fat milk or BSA in TBST (BSA preferred for phospho-detection)

• Antibody incubation:

  • Primary antibody: Dilute 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C for maximum sensitivity

  • Secondary antibody: Anti-rabbit HRP at 1:5000-1:10000, room temperature for 1 hour

• Detection considerations:

  • ECL substrates are suitable for standard detection

  • For low abundance samples, consider enhanced chemiluminescence systems

• Special considerations:

  • When studying DNA damage response, compare untreated vs. irradiated samples

  • For IR-treated samples, collect at multiple timepoints (0h, 0.5h, 2h, 6h post-irradiation) to observe phosphorylation dynamics

How should I optimize immunohistochemistry protocols for PPP4R2 detection in tissue samples?

For successful PPP4R2 detection in tissue samples by IHC:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections at 4-6 μm thickness

  • Validated tissues: Human liver cancer tissue and human pancreas tissue

• Antigen retrieval optimization:

  • Primary recommended method: Heat-induced epitope retrieval with TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Optimize retrieval time (typically 10-20 minutes)

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with 5-10% normal serum from secondary antibody host

  • Primary antibody dilution: 1:20-1:200 in blocking buffer

  • Incubation time: Overnight at 4°C for maximum sensitivity

• Detection and visualization:

  • HRP-conjugated secondary antibody with DAB substrate provides good contrast

  • Counter-stain with hematoxylin for nuclear visualization

  • For co-localization studies, consider fluorescent secondary antibodies

• Controls and validation:

  • Positive control: Include known positive tissue (liver cancer or pancreas)

  • Negative control: Omit primary antibody

  • For tumor studies, compare expression between tumor and adjacent normal tissue

What are the key considerations when using PPP4R2 antibodies for studying DNA damage response mechanisms?

When designing experiments to study PPP4R2's role in DNA damage response:

• Experimental design considerations:

  • Include time-course analysis (0h, 0.5h, 2h, 6h post DNA damage induction)

  • Test multiple DNA damage inducers (IR, UV, chemical agents) for comprehensive analysis

  • Compare wild-type to PPP4R2-depleted cells to establish functional relationships

• Downstream phosphorylation targets to monitor:

  • pKAP1 (S824): Critical DDR protein regulated by PPP4R2

  • γH2AX (S139): DNA damage marker elevated in PPP4R2-deficient cells

  • pRPA2: DNA repair protein affected by PPP4R2 depletion

  • pP53 (S15): Activated in response to PPP4R2 knockdown

• Flow cytometry approach:

  • For high-throughput analysis of DDR protein phosphorylation

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100 or methanol

  • Use antibodies against pKAP1 and γH2AX with appropriate controls

• Western blot considerations:

  • Include phospho-specific antibodies for DDR proteins

  • Monitor both phosphorylated and total protein levels

  • Collect samples at multiple timepoints after IR treatment (2 Gy recommended dose)

• Functional assays to correlate with PPP4R2 status:

  • Homologous recombination (HR) repair efficiency

  • Non-homologous end-joining (NHEJ) pathway activity

  • Cell cycle checkpoint activation

  • Colony formation assays to assess cell survival post-damage

How can I effectively use PPP4R2 antibodies to investigate its role in cancer progression, particularly in hematological malignancies?

For investigating PPP4R2's role in cancer, especially in hematological malignancies:

• Expression analysis strategies:

  • Compare PPP4R2 protein levels between patient samples and healthy controls using western blot or IHC

  • Correlate protein levels with clinical outcomes and genetic alterations

  • Focus on AML patients with 3p deletions or complex karyotypes who show significantly reduced PPP4R2 expression

• Functional investigation approaches:

  • Create isogenic cell line models using RNAi or CRISPR/Cas9 to modulate PPP4R2 levels

  • Assess DDR capacity using γH2AX and pKAP1 as markers

  • Investigate cell proliferation, colony formation, and apoptotic response

  • Examine genomic stability in PPP4R2-deficient cells

• Co-IP approaches to identify PPP4R2 interactors in cancer contexts:

  • Use PPP4R2 antibodies for immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Identify cancer-specific interaction partners

  • Validate in hematological cell lines like K562 or primary AML samples

• In vivo model considerations:

  • Use patient-derived xenografts with varying PPP4R2 status

  • Assess tumor progression and therapy response

  • Perform IHC on resulting tumors to correlate PPP4R2 levels with outcomes

• Transcriptomic correlation analysis:

  • Identify PPP4R2 co-expressed genes by comparing high vs. low PPP4R2-expressing tumors

  • Look for enrichment in DNA repair pathways and cancer-related processes

  • Connect findings to AML pathogenesis mechanisms

What methodological approaches are recommended for studying interactions between PPP4R2 and the DNA damage response pathway components?

For studying PPP4R2 interactions with DDR components:

• Co-immunoprecipitation (Co-IP) strategy:

  • Use PPP4R2 antibodies to pull down protein complexes

  • Optimal antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Compare complexes from untreated vs. DNA-damaged cells

  • HepG2 cells have been validated for PPP4R2 IP

• Proximity ligation assay (PLA) approach:

  • Visualize direct protein-protein interactions in situ

  • Combine PPP4R2 antibody with antibodies against DDR proteins (KAP1, H2AX, RPA2)

  • Quantify interaction signals before and after DNA damage induction

• Mass spectrometry-based interactome analysis:

  • Perform IP with PPP4R2 antibodies followed by MS analysis

  • Compare interactome changes following DNA damage

  • Validate key interactions with reciprocal Co-IP

• Phosphorylation dynamics analysis:

  • Monitor dephosphorylation of known substrates (pKAP1, γH2AX, pRPA2)

  • Establish temporal relationship between PPP4R2 recruitment and substrate dephosphorylation

  • Use phospho-specific antibodies in combination with PPP4R2 antibodies

• Chromatin immunoprecipitation (ChIP)-based approaches:

  • Determine if PPP4R2 is recruited to DNA damage sites

  • Combine with γH2AX ChIP to correlate with damage sites

  • Use sequential ChIP to identify co-recruitment with other DDR factors

How can RNAi-based functional studies of PPP4R2 be designed to ensure specificity and interpretable results?

For designing rigorous RNAi-based PPP4R2 functional studies:

• ShRNA design and validation strategy:

  • Design multiple shRNAs targeting different regions of PPP4R2 mRNA

  • Based on published studies, consider validated constructs like Ppp4r2-sh3 and Ppp4r2-sh4

  • Include non-targeting control shRNA

  • Validate knockdown efficiency by qRT-PCR and western blot using PPP4R2 antibodies

• Functional readout selection:

  • DNA damage markers: γH2AX and pKAP1 levels by flow cytometry and western blot

  • Colony formation capacity of hematopoietic progenitor cells

  • Cell cycle progression and checkpoint activation

  • Genomic stability markers

• Rescue experiment design:

  • Express shRNA-resistant PPP4R2 constructs

  • Confirm expression by western blot with PPP4R2 antibodies

  • Demonstrate restoration of normal DDR signaling

  • Use both wild-type and mutant rescue constructs to identify critical domains

• Controls for off-target effects:

  • Use multiple shRNAs with similar phenotypes

  • Validate key findings with CRISPR/Cas9 knockout

  • Perform transcriptome analysis to identify potential off-target effects

  • Include rescue experiments as described above

• Temporal considerations:

  • Assess acute vs. chronic PPP4R2 depletion effects

  • For DNA damage studies, collect samples at multiple timepoints post-IR (0h, 0.5h, 2h, 6h)

  • Monitor long-term genomic stability in constitutive vs. inducible knockdown systems

What are common technical issues when working with PPP4R2 antibodies and how can they be resolved?

Common technical challenges and solution strategies include:

• Western blot detection issues:

  • Problem: Weak or absent signal

  • Solutions:

    • Increase antibody concentration (try 1:500 instead of 1:2000)

    • Extend primary antibody incubation to overnight at 4°C

    • Use enhanced chemiluminescence detection systems

    • Verify protein extraction efficiency and integrity

• Multiple bands in western blot:

  • Problem: Bands at unexpected molecular weights

  • Solutions:

    • Note that the observed MW (65 kDa) differs from calculated MW (40-55 kDa)

    • Use positive control lysates (A549, HepG2 cells)

    • Include PPP4R2 knockdown controls to identify specific bands

    • Optimize blocking conditions to reduce non-specific binding

• High background in IHC/IF:

  • Problem: Non-specific staining obscuring specific signal

  • Solutions:

    • Optimize antibody dilution (try 1:100-1:200 range)

    • Extend blocking step (10% normal serum, 1-2 hours)

    • Include 0.1-0.3% Triton X-100 in antibody diluent

    • Test alternative antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Immunoprecipitation inefficiency:

  • Problem: Poor target protein enrichment

  • Solutions:

    • Adjust antibody amount (0.5-4.0 μg per 1-3 mg lysate is recommended)

    • Pre-clear lysates to reduce non-specific binding

    • Optimize lysis buffer composition for protein complex preservation

    • Consider crosslinking antibody to beads for cleaner elution

• Antibody cross-reactivity concerns:

  • Problem: Potential non-specific detection

  • Solutions:

    • Validate with multiple antibodies targeting different epitopes

    • Include genetic knockout/knockdown controls

    • Perform peptide competition assays

    • Test species cross-reactivity if working with non-human models

How can I reconcile discrepancies between PPP4R2 antibody detection and other gene expression data?

When facing discrepancies between antibody-based detection and other data:

• Transcript vs. protein level discrepancies:

  • Investigation approach:

    • Verify transcript measurements with multiple primer sets

    • Use multiple antibodies targeting different epitopes of PPP4R2

    • Consider post-transcriptional regulation mechanisms

    • Check for protein degradation in your sample preparation

• Conflicting functional outcomes:

  • Resolution strategy:

    • Design complementary approaches (RNAi, CRISPR, overexpression)

    • Measure outcomes with multiple methodologies

    • Consider cell type-specific effects (PPP4R2 expression varies across cell types)

    • Validate in multiple model systems

• Observed vs. calculated molecular weight discrepancies:

  • Analytical approach:

    • The observed MW of PPP4R2 is 65 kDa, while calculated MWs range from 40-55 kDa

    • Consider post-translational modifications

    • Validate band identity with knockdown controls

    • Check for alternative isoforms (PPP4R2 has 360aa/40kDa and 495aa/55kDa forms)

• Species-specific differences:

  • Management approach:

    • Verify antibody species reactivity (human vs. mouse vs. other models)

    • Compare sequence homology in the epitope region

    • Use species-matched positive controls

    • Consider species-specific alternative splicing or modifications

• Phosphorylation dynamics discrepancies:

  • Reconciliation approach:

    • Use multiple timepoints after stimulation (0h, 0.5h, 2h, 6h post-IR)

    • Compare results from different detection methods (western blot vs. flow cytometry)

    • Control for phosphatase activity during sample preparation

    • Consider context-dependent signaling differences

What emerging roles of PPP4R2 beyond DNA damage response should researchers consider investigating?

Beyond DDR, researchers should consider investigating these emerging PPP4R2 functions:

• Hematopoietic differentiation regulation:

  • Research context: PPP4R2 expression is significantly higher in primitive hematopoietic cells compared to differentiated cells

  • Investigative approach:

    • Track PPP4R2 protein levels during hematopoietic differentiation using western blot

    • Perform lineage-specific knockdown experiments

    • Analyze differentiation marker expression in PPP4R2-depleted models

    • Correlate with hematological disease phenotypes

• Tumor suppressor mechanisms in AML:

  • Research context: PPP4R2 is deleted in a subset of AML patients with 3p deletion or complex karyotype

  • Methodological approach:

    • Compare PPP4R2 protein levels in AML subtypes using antibody-based detection

    • Investigate correlation with specific genetic lesions

    • Examine effects of PPP4R2 restoration in deficient cell lines

    • Analyze therapeutic vulnerability in PPP4R2-deficient tumors

• Wnt signaling pathway interactions:

  • Research context: PPP4C (catalytic subunit partner of PPP4R2) has been identified as a Wnt regulator

  • Experimental design:

    • Investigate if PPP4R2 mediates PPP4C effects on Wnt signaling

    • Use Co-IP to identify interactions with Wnt pathway components

    • Analyze β-catenin activity in PPP4R2-modulated systems

    • Correlate with developmental and cancer phenotypes

• Cell cycle regulation:

  • Research context: PPP4R2 impacts DNA damage checkpoints

  • Investigation strategy:

    • Analyze cell cycle profiles in PPP4R2-depleted cells before/after DNA damage

    • Use PPP4R2 antibodies to track subcellular localization during cell cycle phases

    • Identify cell cycle-regulated PPP4R2 phosphorylation sites

    • Examine interactions with centrosomal proteins during mitosis

• Therapeutic targeting potential:

  • Research context: PPP4R2 status may influence therapy response

  • Experimental approach:

    • Screen drug sensitivity in PPP4R2-proficient vs. deficient models

    • Analyze synergy with DNA-damaging agents

    • Investigate synthetic lethality relationships

    • Use PPP4R2 antibodies to monitor protein levels during treatment response

How can researchers integrate PPP4R2 antibody-based techniques with other advanced methodologies for comprehensive functional studies?

Integration strategies for comprehensive PPP4R2 functional studies:

• Combination with CRISPR-based genome editing:

  • Create precise PPP4R2 knockouts or domain mutants

  • Validate with PPP4R2 antibodies

  • Compare phenotypes with RNAi-based approaches

  • Create tagged endogenous PPP4R2 for live imaging

• Integration with phosphoproteomics:

  • Compare global phosphorylation changes in PPP4R2-proficient vs. deficient cells

  • Focus on DDR-related phosphosites (KAP1, H2AX, RPA2)

  • Validate key findings with phospho-specific antibodies

  • Construct signaling networks centered on PPP4R2

• Single-cell analysis integration:

  • Combine with single-cell RNA-seq to identify cell state-specific effects

  • Use IF with PPP4R2 antibodies for spatial analysis in tissues

  • Correlate protein levels with cellular phenotypes

  • Analyze heterogeneity in PPP4R2 expression and function

• Proximity labeling methodologies:

  • Fuse BioID or APEX2 to PPP4R2

  • Map protein-protein interactions in different cellular compartments

  • Compare interactome before/after DNA damage

  • Validate key interactions with co-IP using PPP4R2 antibodies

• In vivo imaging and lineage tracing:

  • Use PPP4R2 antibodies for tissue analyses in developmental studies

  • Track PPP4R2-dependent processes in disease models

  • Combine with functional readouts (proliferation, apoptosis, differentiation)

  • Correlate with genetic manipulation studies

What considerations should researchers take into account when designing experiments to study PPP4R2's role in different cancer subtypes?

When investigating PPP4R2 across cancer subtypes:

• Cancer-specific expression pattern analysis:

  • Experimental design:

    • Survey PPP4R2 protein levels across cancer types using antibody-based detection

    • Compare with matched normal tissues

    • Correlate with genomic data (deletion status, mutations)

    • Pay special attention to AML with 3p deletions and complex karyotypes

• Functional significance assessment:

  • Methodological approach:

    • Modulate PPP4R2 levels in multiple cancer cell lines

    • Compare phenotypic outcomes across lineages

    • Assess DDR efficiency using pKAP1 and γH2AX markers

    • Evaluate cancer-specific synthetic lethality relationships

• Clinical correlation strategy:

  • Research design:

    • Use PPP4R2 antibodies for IHC on tissue microarrays

    • Correlate protein levels with patient outcomes

    • Analyze therapy response data in relation to PPP4R2 status

    • Consider PPP4R2 as a biomarker for specific treatments

• Cancer subtype-specific pathway analysis:

  • Investigation approach:

    • Identify PPP4R2-dependent pathways in different cancer contexts

    • Use Co-IP to detect cancer-specific interaction partners

    • Perform transcriptome analysis of PPP4R2-modulated cancer cells

    • Connect findings to known cancer driver pathways

• Therapeutic vulnerability screening:

  • Experimental strategy:

    • Test drug sensitivity in PPP4R2-deficient vs. proficient models

    • Focus on DNA-damaging agents due to PPP4R2's role in DDR

    • Screen for synthetic lethal interactions

    • Validate findings across multiple cancer subtypes

• Microenvironment considerations:

  • Research approach:

    • Study PPP4R2 function in co-culture systems

    • Analyze effects of hypoxia and nutrient deprivation on PPP4R2 function

    • Investigate immune response correlation with PPP4R2 status

    • Use PPP4R2 antibodies to study expression in tumor-associated cells