PPP2R5E Antibody

What is PPP2R5E and what cellular functions does it regulate?

PPP2R5E (Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform) is a regulatory B' subunit of protein phosphatase 2A (PP2A). It modulates substrate selectivity and catalytic activity of the phosphatase complex while directing the localization of the catalytic enzyme to specific subcellular compartments . Recent research has demonstrated that PPP2R5E interacts with microtubule crosslinking factor 1 (MTCL1) and contributes to microtubule organization by stabilizing MTCL1 . The depletion of PPP2R5E reduces MTCL1 abundance, while exogenous expression increases endogenous MTCL1 levels, indicating a regulatory relationship between these proteins . Additionally, cells depleted of PPP2R5E exhibit defects in microtubule organization, highlighting its importance in cytoskeletal dynamics .

What are the common applications for PPP2R5E antibodies in research?

PPP2R5E antibodies are utilized across multiple laboratory techniques to study protein expression, localization, and interactions. The primary applications include:

• Western blotting (WB): For detection and quantification of PPP2R5E protein levels

• Immunoprecipitation (IP): To isolate PPP2R5E and its binding partners

• Immunohistochemistry (IHC-P): For visualization in paraffin-embedded tissues

• Immunocytochemistry/Immunofluorescence (ICC/IF): To examine subcellular localization

• Flow cytometry: For intracellular detection in cell populations

When selecting an antibody, researchers should verify which applications have been validated for each specific antibody, as performance can vary considerably between different experimental techniques and conditions.

How should I select the appropriate PPP2R5E antibody for my experiment?

Selection of the appropriate PPP2R5E antibody requires consideration of several factors:

• Target epitope: Different antibodies target different regions of PPP2R5E, such as N-terminal (AA 2-149), C-terminal, or specific internal domains (AA 35-84, AA 120-169, etc.)

• Host species: Consider rabbit polyclonal or monoclonal antibodies based on your experimental design and secondary antibody compatibility

• Clonality: Monoclonal antibodies provide higher specificity but potentially lower sensitivity compared to polyclonal antibodies

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IP, IHC, etc.)

• Cross-reactivity: Check species cross-reactivity if working with non-human models

• Conjugation: Determine if you need unconjugated or conjugated (e.g., HRP) formats

For critical experiments, it's advisable to test multiple antibodies targeting different epitopes to confirm specificity and reproducibility of results.

What controls should I include when using PPP2R5E antibodies?

Proper controls are essential for interpreting PPP2R5E antibody results:

The gold standard for antibody validation includes comparing signals between wild-type and knockout cell lines, which provides definitive evidence of specificity . When possible, using multiple antibodies targeting different epitopes can further strengthen confidence in your results.

How can I optimize Western blot protocols for PPP2R5E detection?

Optimizing Western blot protocols for PPP2R5E detection requires addressing several technical considerations:

• Sample preparation:

  • Use phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • Optimize protein extraction by testing different lysis buffers (RIPA vs. NP-40)

  • Consider subcellular fractionation, as PPP2R5E localizes to specific compartments

• Electrophoresis conditions:

  • PPP2R5E has a molecular weight of approximately 55 kDa

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Consider gradient gels if detecting PPP2R5E complexes

• Transfer optimization:

  • Semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 100V for 60-90 minutes

  • Use PVDF membranes for better protein retention and signal-to-noise ratio

• Blocking and antibody incubation:

  • Test different blocking agents (5% BSA often works better than milk for phosphoproteins)

  • Optimal primary antibody dilution typically ranges from 1:500 to 1:2000

  • Incubate primary antibody overnight at 4°C for improved sensitivity

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) for standard detection

  • Consider fluorescent secondary antibodies for multiplex detection and quantification

For challenging samples, signal enhancement systems or high-sensitivity substrates may improve detection of low-abundance PPP2R5E protein .

What are the common challenges in PPP2R5E immunoprecipitation experiments and how can they be addressed?

Immunoprecipitation (IP) of PPP2R5E presents several challenges:

• Antibody selection: Not all antibodies perform well in IP applications. Validate using known positive controls and compare efficiency between different antibodies .

• Preserving protein interactions:

  • Use gentle lysis buffers (e.g., 0.5% NP-40 with physiological salt concentrations)

  • Include phosphatase inhibitors to maintain native phosphorylation states

  • Consider crosslinking before lysis for transient interactions

• Background reduction:

  • Pre-clear lysates with Protein A/G beads

  • Use specific monoclonal antibodies to reduce non-specific binding

  • Optimize wash stringency (salt concentrations, detergent levels)

• Validation of results:

  • Perform reciprocal IPs with antibodies against interacting partners

  • Include appropriate negative controls (isotype control, IgG, knockout samples)

  • Confirm specificity with Western blot detection of immunoprecipitated material

• Detecting low-abundance complexes:

  • Scale up starting material

  • Use sensitive detection methods for Western blotting

  • Consider mass spectrometry for unbiased identification of interacting partners

For studying PPP2R5E's interaction with MTCL1, maintaining phosphorylation status is particularly important as their interaction appears to be phosphorylation-dependent .

How can I effectively validate PPP2R5E antibody specificity in my experimental system?

Comprehensive validation of PPP2R5E antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Compare signal between wild-type and PPP2R5E knockout cell lines

  • Use siRNA or shRNA knockdown to create partial depletion controls

  • Perform rescue experiments with exogenous PPP2R5E expression

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Observe signal reduction in peptide-blocked samples

• Orthogonal detection methods:

  • Compare results using antibodies targeting different epitopes

  • Verify with orthogonal techniques (mass spectrometry, RNA expression)

• Cross-reactivity assessment:

  • Test against related family members (other B56 subunits)

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

• Application-specific validation:

  • For immunofluorescence: Compare staining patterns with multiple antibodies

  • For Western blot: Verify band size and compare with predicted molecular weight

  • For IP: Confirm pull-down efficiency and specificity

The mosaic approach, where wild-type and knockout cells are labeled with different fluorescent dyes and imaged in the same field of view, provides a robust method for validating antibodies for immunofluorescence applications .

What is the significance of PPP2R5E's interaction with MTCL1 and how can I study this interaction?

PPP2R5E's interaction with microtubule crosslinking factor 1 (MTCL1) represents a critical regulatory mechanism in microtubule organization. This interaction has several important implications:

• Functional significance:

  • PPP2R5E stabilizes MTCL1 through dephosphorylation

  • Depletion of PPP2R5E reduces MTCL1 abundance

  • Cells lacking either protein exhibit defects in microtubule organization

• Experimental approaches to study the interaction:

  • Co-immunoprecipitation: Pull down PPP2R5E and probe for MTCL1, or vice versa

  • Proximity ligation assay: Visualize protein-protein interactions in situ

  • FRET/BRET: Measure direct protein interactions in living cells

  • Yeast two-hybrid: Map interaction domains

• Phosphorylation dynamics:

  • Okadaic acid treatment (phosphatase inhibitor) reduces MTCL1 levels

  • This effect can be rescued by proteasome inhibitor MG132, suggesting phosphorylation regulates MTCL1 stability

  • Phospho-specific antibodies can help track MTCL1 phosphorylation status

• Cellular consequences:

  • Immunofluorescence microscopy to visualize microtubule organization

  • Live-cell imaging to track microtubule dynamics

  • Functional assays for processes dependent on proper microtubule organization

When designing experiments to study this interaction, consider that phosphatase activity inhibition by okadaic acid reduces MTCL1 levels, which can be restored by adding protease inhibitor MG132, indicating phosphorylation-dependent proteolytic regulation .

How do I troubleshoot inconsistent results when using PPP2R5E antibodies for immunofluorescence?

Inconsistent immunofluorescence results with PPP2R5E antibodies can result from various factors:

• Fixation method optimization:

  • Compare paraformaldehyde vs. methanol fixation

  • Test different fixation durations (10-20 minutes)

  • For membrane proteins, avoid harsh permeabilization

• Antibody optimization:

  • Titrate antibody concentration (typically 1:100 to 1:500)

  • Test different incubation times and temperatures

  • Compare different antibodies targeting distinct epitopes

• Signal-to-noise improvement:

  • Increase blocking time or concentration (5% BSA or 10% normal serum)

  • Add 0.1-0.3% Triton X-100 for better permeabilization

  • Include multiple washing steps with 0.1% Tween-20

• Antigen retrieval considerations:

  • For fixed tissues, test heat-induced or enzymatic antigen retrieval

  • Optimize pH of retrieval buffers (citrate buffer pH 6.0 vs. Tris-EDTA pH 9.0)

• Specificity validation:

  • Use the mosaic approach with wild-type and knockout cells

  • Include peptide competition controls

  • Compare with other subcellular markers to confirm localization pattern

• Image acquisition settings:

  • Standardize exposure settings between experiments

  • Use appropriate negative controls to set background thresholds

  • Consider spectral unmixing for multi-color experiments

Quantification of immunofluorescence intensity in hundreds of wild-type and knockout cells provides robust statistical validation of antibody specificity and can help identify optimal staining conditions .

How can I distinguish between different PPP2R5 family members in my experiments?

Distinguishing between the five PPP2R5 isoforms (α, β, γ, δ, and ε) requires careful experimental design:

• Antibody selection:

  • Use antibodies targeting unique regions that differ between isoforms

  • Validate specificity against recombinant proteins of each isoform

  • Consider custom antibodies for highly specific detection

• Experimental approaches:

  • Western blotting: Different isoforms have slightly different molecular weights

  • RT-qPCR: Design primers specific to unique regions of each isoform

  • Mass spectrometry: Identify isoform-specific peptides

• Genetic manipulation:

  • Use isoform-specific siRNA/shRNA

  • Create knockout cell lines for individual isoforms

  • Perform rescue experiments with individual isoforms

• Cross-validation:

  • Compare protein data with RNA expression data

  • Use publicly available tissue expression databases to predict predominant isoforms

• Functional differentiation:

  • Design assays that detect isoform-specific functions

  • Identify isoform-specific binding partners

The B' family members share 56-72% sequence identity, making specificity particularly important. When interpreting results, consider the relative expression levels of different isoforms in your experimental system, as this can affect interpretation of functional studies .

What are the considerations for analyzing PPP2R5E in different subcellular compartments?

PPP2R5E can localize to different subcellular compartments, requiring specialized approaches for comprehensive analysis:

• Subcellular fractionation techniques:

  • Differential centrifugation to separate major organelles

  • Density gradient separation for higher resolution

  • Digitonin permeabilization for cytosolic vs. membrane-bound separation

• Immunofluorescence co-localization:

  • Use established markers for different compartments (nucleus, cytosol, mitochondria, etc.)

  • Employ super-resolution microscopy for precise localization

  • Quantify co-localization using Pearson's or Manders' coefficients

• Biochemical considerations:

  • Different extraction buffers may be required for different compartments

  • Use compartment-specific markers as controls (GAPDH for cytosol, Histone H3 for nucleus)

  • Consider phosphorylation status, which may differ between compartments

• Functional context:

  • The B regulatory subunit directs the localization of the catalytic enzyme to particular subcellular compartments

  • Different compartments may contain different PPP2R5E binding partners

  • Consider cell-cycle or stimulus-dependent changes in localization

• Quantification approaches:

  • Measure relative distribution across compartments

  • Track changes in localization following specific stimuli

  • Consider dynamic behaviors using live-cell imaging

When analyzing results, remember that PPP2R5E's role in directing PP2A activity to specific subcellular locations is a key aspect of its regulatory function .

How should I design experiments to study PPP2R5E's role in microtubule organization?

Designing experiments to study PPP2R5E's role in microtubule organization requires multiple complementary approaches:

• Genetic manipulation strategies:

  • CRISPR/Cas9 knockout of PPP2R5E

  • siRNA/shRNA knockdown for partial depletion

  • Expression of dominant-negative mutants

  • Rescue experiments with wild-type or mutant PPP2R5E

• Microscopy approaches:

  • Immunofluorescence to visualize microtubule structure

  • Live-cell imaging with fluorescently-tagged tubulin

  • Super-resolution microscopy for detailed structural analysis

  • Quantitative image analysis of microtubule organization parameters

• Biochemical assays:

  • Microtubule co-sedimentation to assess direct interactions

  • Analysis of tubulin post-translational modifications

  • Assessment of microtubule stability (resistance to cold or nocodazole)

  • Co-immunoprecipitation of PPP2R5E with MTCL1 and tubulin

• Functional readouts:

  • Microtubule regrowth after nocodazole washout

  • Cell migration assays (wound healing, transwell)

  • Cell division analysis (spindle formation, chromosome segregation)

  • Neurite outgrowth in neuronal models

• Phosphorylation dynamics:

  • Phosphatase inhibition with okadaic acid

  • Proteasome inhibition with MG132

  • Analysis of MTCL1 phosphorylation status

Research has shown that cells depleted of PPP2R5E exhibit defects in microtubule organization, similar to the defects observed in MTCL1-depleted cells, suggesting PPP2R5E contributes to microtubule organization by stabilizing MTCL1 .

What methodological approaches can help resolve contradictory data about PPP2R5E function?

Resolving contradictory data about PPP2R5E function requires systematic troubleshooting and experimental design:

• Validate reagents:

  • Test multiple antibodies targeting different epitopes

  • Verify knockout/knockdown efficiency using multiple methods

  • Consider epitope accessibility issues in different experimental contexts

• Address context-dependency:

  • Test in multiple cell lines to identify cell-type specific effects

  • Consider cell cycle stage and growth conditions

  • Evaluate acute vs. chronic depletion effects

• Dose-dependency analysis:

  • Use titrated knockdown approaches (variable siRNA concentrations)

  • Create dose-response curves for inhibitor treatments

  • Employ inducible expression systems for controlled protein levels

• Time-course experiments:

  • Track changes over time after manipulation

  • Consider adaptive responses to protein depletion

  • Distinguish between direct and secondary effects

• Control for off-target effects:

  • Use multiple siRNA sequences

  • Include rescue experiments with siRNA-resistant constructs

  • Employ CRISPR knockout with multiple guide RNAs

• Orthogonal approaches:

  • Combine genetic, biochemical, and imaging methods

  • Use different experimental systems (in vitro, cellular, in vivo)

  • Compare with published data on related family members

When facing contradictory results, consider that PPP2R5E functions through protein-protein interactions that may be influenced by cell type, protein expression levels, and experimental conditions .

How can advanced proteomic approaches enhance PPP2R5E research?

Advanced proteomic approaches offer powerful tools for comprehensive PPP2R5E research:

• Interactome analysis:

  • Affinity purification-mass spectrometry (AP-MS)

  • Proximity-dependent biotin identification (BioID)

  • Cross-linking mass spectrometry (XL-MS) for structural insights

  • Co-immunoprecipitation followed by liquid chromatography-mass spectrometry (LC-MS/MS)

• Post-translational modification mapping:

  • Phosphoproteomic analysis of PPP2R5E substrates

  • Identification of PPP2R5E's own modification sites

  • Quantitative analysis of phosphorylation changes upon PPP2R5E manipulation

• Targeted proteomics approaches:

  • Selected reaction monitoring (SRM) for absolute quantification

  • Parallel reaction monitoring (PRM) for improved selectivity

  • AQUA peptides for standardized quantification

• Structural proteomics:

  • Hydrogen-deuterium exchange (HDX) for conformational analysis

  • Native mass spectrometry for complex integrity

  • Integrative structural biology combining multiple methods

• Spatial proteomics:

  • Imaging mass spectrometry for tissue localization

  • Subcellular fractionation coupled with proteomics

  • In situ proximity labeling for compartment-specific interactomes

• Dynamic proteomics:

  • Pulse-chase SILAC for protein turnover rates

  • Thermal proteome profiling for stability changes

  • Time-resolved interactome studies

These advanced approaches can help resolve contradictory findings by providing unbiased, comprehensive data on PPP2R5E function, interactions, and regulation across different experimental contexts.