PP2B11 Antibody

What is PP2B11 and what cellular functions does it regulate?

PP2B11 (also known as SKIP12, At1g80110, F18B13.19, F-box protein PP2-B11, Protein PHLOEM PROTEIN 2-LIKE B11, AtPP2-B11, or SKP1-interacting partner 12) functions as a key component of SCF (SKP1-cullin-F-box) E3 ubiquitin ligase complexes. These complexes play essential roles in cellular protein homeostasis by mediating the ubiquitination and subsequent proteasomal degradation of target proteins. In plants, particularly Arabidopsis, PP2B11 has been demonstrated to specifically regulate abscisic acid (ABA) signaling pathways through targeted degradation of SnRK2.3, effectively attenuating ABA responses and abiotic stress response mechanisms. This regulatory function positions PP2B11 as a critical negative regulator in plant stress response systems, making it an important target for agricultural and environmental stress research.

How does PP2B11 contribute to plant stress responses?

PP2B11 plays a significant role in modulating plant responses to environmental stressors, particularly through its involvement in ABA signaling pathways. Research has demonstrated that PP2B11 expression is induced by ABA treatment. Functional studies using genetic approaches have revealed that knockdown of AtPP2-B11 expression significantly increases plant sensitivity to ABA during critical developmental stages, including seed germination and post-germinative development. This suggests PP2B11 normally functions to attenuate ABA signaling. Additionally, transgenic Arabidopsis plants engineered to overexpress AtPP2-B11 exhibited pronounced tolerance to high salinity conditions, whereas RNA interference lines with reduced PP2B11 expression demonstrated increased sensitivity to salt stress compared to wild-type plants. These findings collectively indicate that PP2B11 functions as a regulatory component in stress response pathways, specifically through modulation of ABA signaling by targeting SnRK2.3 for degradation.

What experimental systems are most appropriate for studying PP2B11 function?

The optimal experimental systems for investigating PP2B11 function are plant models, particularly Arabidopsis thaliana, where the protein's role has been most extensively characterized. When designing experiments, researchers should consider:

• Genetic manipulation approaches:

  • Overexpression lines to assess gain-of-function phenotypes

  • RNA interference (RNAi) or CRISPR-Cas9 knockout lines to evaluate loss-of-function effects

  • Complementation studies using mutant lines expressing wild-type or modified PP2B11 variants

• Stress treatment conditions:

  • ABA application experiments (typically 0.5-50 μM range)

  • Salt stress protocols (commonly using NaCl at concentrations of 100-200 mM)

  • Drought stress simulations using controlled soil moisture conditions or polyethylene glycol treatments

• Developmental contexts:

  • Seed germination assays

  • Post-germinative growth assessment

  • Adult plant response monitoring

Since PP2B11 belongs to the phloem protein family, phloem-specific experimental approaches may also prove valuable, particularly when investigating tissue-specific functions .

What are the key applications for PP2B11 antibodies in research?

PP2B11 antibodies serve multiple critical applications in plant molecular biology and stress physiology research:

• Protein expression analysis: Western blotting to detect and quantify native PP2B11 protein levels across different tissues, developmental stages, or in response to various stress conditions.

• Protein localization studies: Immunofluorescence or immunohistochemistry to determine the subcellular and tissue-specific localization patterns of PP2B11, particularly in phloem tissues.

• Protein-protein interaction investigations: Immunoprecipitation (IP) assays to identify interaction partners within the SCF complex or with target proteins like SnRK2.3.

• Chromatin immunoprecipitation (ChIP): If PP2B11 has any DNA-binding or chromatin-associated functions, though current evidence suggests its primary role is in protein degradation pathways.

• ELISA-based quantification: For precise measurement of PP2B11 protein levels in plant extracts under various experimental conditions.

These applications require properly validated antibodies with confirmed specificity against the target protein.

How can I validate PP2B11 antibody specificity in my experimental system?

Validating antibody specificity is crucial for obtaining reliable results. For PP2B11 antibodies, implement the following comprehensive validation strategy:

• Western blot analysis with appropriate controls:

  • Compare wild-type samples with PP2B11 overexpression lines (positive control)

  • Include PP2B11 knockout/knockdown lines (negative control)

  • Analyze tissue types with known differential expression patterns

  • Verify that the detected band appears at the expected molecular weight (~52 kDa for Arabidopsis PP2B11)

• Peptide competition assay:

  • Pre-incubate the antibody with excess purified PP2B11 protein or immunizing peptide

  • Compare results with non-competed antibody detection

  • Signal reduction/elimination confirms specificity

• Cross-reactivity assessment:

  • Test against closely related PP2 family members (especially PP2-B13, which has similar structure)

  • Validate against heterologously expressed PP2B11 with epitope tags

  • Examine potential cross-reactivity with SCF complex components

• Immunoprecipitation validation:

  • Confirm that immunoprecipitated proteins include known PP2B11 interaction partners (e.g., SKP1)

  • Verify enrichment of PP2B11 by mass spectrometry analysis

Researchers should note that PP2B11 antibody buffer typically contains 0.03% Proclin 300 as a preservative and is formulated in 50% glycerol with 0.01M PBS at pH 7.4, which may influence some validation procedures.

What methodological approaches can identify novel PP2B11 substrates beyond SnRK2.3?

To discover novel substrates of PP2B11 beyond the known target SnRK2.3, researchers should employ multiple complementary approaches:

• Proximity-based protein identification:

  • BioID or TurboID fusion protein expression followed by streptavidin pulldown and mass spectrometry

  • APEX2-based proximity labeling combined with proteomics

  • These methods can capture transient interactions typical of enzyme-substrate relationships

• Quantitative proteomics approaches:

  • Compare ubiquitinated protein profiles between wild-type and PP2B11 mutant plants using diGly-Lys enrichment and mass spectrometry

  • Implement stable isotope labeling (SILAC or TMT) to quantify protein abundance changes

  • Focus analysis on proteins with increased stability in PP2B11-deficient backgrounds

• Candidate-based interaction testing:

  • Screen proteins within ABA signaling or salt stress response pathways

  • Test direct interactions using yeast two-hybrid or split-luciferase complementation assays

  • Validate via co-immunoprecipitation using PP2B11 antibodies

• Domain-focused approaches:

  • Identify proteins containing motifs similar to the degradation sequences in SnRK2.3

  • Generate PP2B11 mutants with altered substrate-binding domains and assess differential impacts

How can I analyze PP2B11's involvement in abiotic stress tolerance mechanisms?

To comprehensively investigate PP2B11's role in abiotic stress tolerance:

• Transcriptomic profiling:

  • Conduct RNA-seq comparing wild-type, PP2B11-overexpressing, and PP2B11-deficient plants under normal and stress conditions (salt, drought, ABA treatment)

  • Analyze differentially expressed gene networks with particular focus on known stress response pathways

  • The high-throughput RNA sequencing approach used in study provides a useful methodological template

• Physiological phenotyping:

  • Measure germination rates under varying concentrations of NaCl (50-200 mM) and ABA (0.1-10 μM)

  • Quantify growth parameters (root length, biomass, leaf area) during stress exposure

  • Assess water loss rates, relative water content, and stomatal conductance

  • Monitor ion accumulation patterns (Na+, K+) in different tissues

• Biochemical analysis:

  • Determine ABA levels using ELISA or LC-MS/MS in different genotypes

  • Measure activities of antioxidant enzymes (SOD, CAT, APX) under stress conditions

  • Quantify stress markers including proline, malondialdehyde, and H₂O₂

• Protein-level investigations:

  • Use PP2B11 antibodies to track protein accumulation during stress exposure

  • Analyze post-translational modifications of PP2B11 that might regulate its activity

  • Examine protein-protein interactions that change under stress conditions

Research has already established that transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibit enhanced tolerance to high salinity, while knockdown lines show increased sensitivity. This provides a solid foundation for more detailed mechanistic studies.

What approaches can I use to study PP2B11's role in ubiquitination pathways?

To investigate PP2B11's function within ubiquitination pathways:

• In vitro ubiquitination assays:

  • Reconstitute the complete SCF^PP2B11 complex using purified components

  • Test ubiquitination activity against known (SnRK2.3) and candidate substrates

  • Analyze ubiquitin chain topology (K48 vs. K63 linkages) using linkage-specific antibodies

  • Include controls with mutated F-box domains to confirm specificity

• Cell-free degradation assays:

  • Prepare plant extracts from wild-type and PP2B11 mutant lines

  • Add purified substrates and monitor their degradation kinetics

  • Test effects of proteasome inhibitors (MG132) and ubiquitin pathway inhibitors

• Structural and functional domain analysis:

  • Generate PP2B11 variants with mutations in key functional domains:

    • F-box domain (substrate recruitment to SCF complex)

    • Substrate recognition domains

    • Potential regulatory domains

  • Express these variants in PP2B11-deficient backgrounds and assess complementation

• Dynamic interaction studies:

  • Use PP2B11 antibodies for time-course immunoprecipitation following stress induction

  • Implement FRET or BiFC approaches to visualize interactions in living cells

  • Apply phosphorylation or other PTM-specific antibodies to monitor modification states of interaction partners

Since PP2B11 is a component of SCF E3 ubiquitin ligase complexes that target proteins for proteasomal degradation, these approaches can help elucidate its specific role within this pathway and identify novel regulatory mechanisms.

How do PP2B11 and related PP2 family members coordinate distinct stress responses?

The PP2 (Phloem Protein 2) family includes multiple members with potentially overlapping and distinct functions. To investigate their coordinated roles:

• Comparative expression analysis:

  • Profile expression patterns of PP2 family members (including PP2-B13, a related protein identified in study ) across tissues and stress conditions

  • Use PP2B11 antibodies alongside antibodies against other family members for protein-level comparisons

  • Implement promoter-reporter fusions to visualize tissue-specific expression patterns

• Higher-order genetic analysis:

  • Generate double, triple, or higher-order mutants disrupting multiple PP2 family members

  • Assess phenotypic consequences under various stress conditions

  • Compare with single mutant phenotypes to identify synergistic, additive, or epistatic relationships

• Substrate specificity determination:

  • Perform comparative interactome studies for different PP2 family proteins

  • Identify shared versus specific interaction partners

  • Conduct competitive binding assays to test substrate preferences

• Evolutionary analysis:

  • Compare PP2 family structure across plant species with varying stress tolerance profiles

  • Identify conserved domains and species-specific adaptations

  • Correlate evolutionary patterns with habitat-specific stressors

Research has already established that PP2-B13, another PP2 family member, is strongly induced in response to flg22 (a bacterial flagellin peptide) treatment, suggesting its involvement in plant immune responses . This contrasts with PP2B11's documented role in abiotic stress tolerance, indicating functional diversification within this protein family that warrants further investigation.

Why might I observe inconsistent results with PP2B11 antibody detection?

Inconsistent PP2B11 antibody detection can stem from several methodological factors:

• Antibody-specific considerations:

  • Storage conditions: PP2B11 antibodies are typically preserved in 0.03% Proclin 300 with 50% glycerol in 0.01M PBS (pH 7.4). Improper storage can lead to antibody degradation

  • Batch variation: Different antibody lots may have varying affinities or specificities

  • Concentration optimization: Each application requires specific antibody dilutions

• Sample preparation factors:

  • Protein extraction efficiency: Different extraction buffers may yield variable PP2B11 recovery

  • Protein stability: PP2B11 may be subject to rapid degradation during sample processing

  • Post-translational modifications: Stress conditions may alter PP2B11's modification state, affecting antibody recognition

• Biological variables:

  • Developmental stage: PP2B11 expression varies across plant development

  • Tissue specificity: As a phloem protein family member, expression may be concentrated in vascular tissues

  • Stress-dependent regulation: ABA and salt stress alter PP2B11 levels

• Technical recommendations:

  • Include positive controls (overexpression lines) and negative controls (knockout lines)

  • Standardize protein extraction using protease inhibitors and denaturing conditions

  • Optimize antibody concentration through titration experiments

  • Consider fixation methods carefully for immunohistochemistry applications

If inconsistent results persist despite optimization, researchers should validate their antibody's specificity against recombinant PP2B11 protein and consider testing alternative antibody sources.

What controls should I include when studying PP2B11 in plant stress responses?

Robust experimental design for studying PP2B11 in stress responses requires comprehensive controls:

• Genetic controls:

  • Wild-type plants (negative control)

  • PP2B11 overexpression lines (gain-of-function control)

  • PP2B11 knockout/knockdown lines (loss-of-function control)

  • Complementation lines (rescue control)

  • Lines with mutations in PP2B11 functional domains (mechanistic controls)

• Treatment controls:

  • Mock treatments (solvent-only controls)

  • Concentration gradients of stressors (dose-response analysis)

  • Time-course experiments (temporal response patterns)

  • Combined stress treatments (stress interaction analysis)

• Molecular controls:

  • SnRK2.3 assessment (known target)

  • Other SCF complex components

  • Related PP2 family members like PP2-B13

  • Known stress response markers

• Control for environmental variables:

  • Standardize growth conditions (light, temperature, humidity)

  • Randomize experimental units

  • Include internal reference genes for qPCR analysis

  • Normalize protein loading with housekeeping proteins

• Antibody controls:

  • Pre-immune serum controls

  • Antibody pre-absorption with immunizing peptide

  • Secondary antibody-only controls

  • Cross-reactivity controls with related proteins

These comprehensive controls help distinguish specific PP2B11-mediated effects from general stress responses or experimental artifacts.

How can I optimize immunoprecipitation protocols for studying PP2B11 interactions?

For effective immunoprecipitation of PP2B11 complexes:

• Extraction buffer optimization:

  • Use mild, non-denaturing conditions: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40 or 1% Triton X-100

  • Include protease inhibitors: PMSF (1 mM), leupeptin (1 μg/ml), aprotinin (1 μg/ml)

  • Add phosphatase inhibitors: sodium fluoride (10 mM), sodium orthovanadate (1 mM)

  • For ubiquitination studies, include deubiquitinase inhibitors: N-ethylmaleimide (5-10 mM)

  • Consider proteasome inhibitors: MG132 (50 μM) to stabilize ubiquitinated substrates

• Antibody coupling strategies:

  • Direct coupling: Crosslink PP2B11 antibodies to protein A/G beads using dimethyl pimelimidate

  • Indirect coupling: Use protein A/G beads to capture antibody-antigen complexes

  • Pre-clearing: Remove non-specific binding proteins with IgG-coupled beads

• Washing conditions:

  • Implement stringency gradient: Start with buffer matching extraction conditions

  • Increase salt concentration (150 mM to 300 mM NaCl) in subsequent washes

  • Final washes with detergent-free buffer

• Elution methods:

  • Competitive elution with excess immunizing peptide (preserves native interactions)

  • Acidic elution: 0.1 M glycine (pH 2.5-3.0) followed by immediate neutralization

  • Denaturing elution: SDS sample buffer with heating (disrupts all interactions)

• Verification approaches:

  • Western blot for known interaction partners (SKP1, Cullins)

  • Mass spectrometry to identify novel components

  • Reverse immunoprecipitation with antibodies against interaction partners

The choice between different approaches depends on whether the goal is to identify stable complex components or capture transient enzyme-substrate interactions.

What factors affect PP2B11 expression and activity in different experimental systems?

Multiple factors influence PP2B11 expression and activity:

• Transcriptional regulators:

  • ABA treatment induces PP2B11 expression

  • Potential involvement of ABA-responsive transcription factors (ABFs, AREBs)

  • Stress-responsive elements in the PP2B11 promoter

• Post-transcriptional regulation:

  • mRNA stability factors

  • Alternative splicing possibilities

  • miRNA regulation potential

• Post-translational modifications:

  • Phosphorylation sites that may regulate PP2B11 activity

  • Potential auto-ubiquitination mechanisms

  • SUMOylation or other modifications that could alter function

• Protein stabilization factors:

  • Interaction partners that might protect PP2B11 from degradation

  • Subcellular localization affecting stability

  • Stress conditions altering protein half-life

• Experimental system considerations:

  • Tissue-specific expression patterns

  • Developmental stage effects

  • Light/dark cycle influences

  • Nutrient availability impacts

Research has shown that transgenic plants overexpressing AtPP2-B11 exhibit enhanced salt tolerance, suggesting that protein levels directly correlate with stress response capacity. Similarly, knockdown of AtPP2-B11 increases ABA sensitivity, indicating that even moderate changes in expression levels can significantly impact phenotypes. Researchers should carefully control these factors when designing experiments to study PP2B11 function.

What are the optimal conditions for using PP2B11 antibodies in immunofluorescence studies?

For successful immunofluorescence detection of PP2B11:

• Sample preparation:

  • Fixation: 4% paraformaldehyde in PBS (pH 7.4) for 20-30 minutes

  • Permeabilization: 0.1-0.3% Triton X-100 for 10-15 minutes

  • Antigen retrieval: Consider mild heat treatment (80°C in citrate buffer, pH 6.0) if initial detection is weak

  • Blocking: 2-5% BSA or normal serum from secondary antibody host species

• Antibody application:

  • Primary antibody: Start with 1:100-1:500 dilution of PP2B11 antibody in blocking solution

  • Incubation: Overnight at 4°C or 2-4 hours at room temperature

  • Secondary antibody: Fluorophore-conjugated anti-species IgG at 1:200-1:1000

  • Nuclear counterstain: DAPI (1 μg/ml) for orientation

• Imaging optimization:

  • Use confocal microscopy for precise subcellular localization

  • Implement appropriate filter sets matching secondary antibody fluorophores

  • Capture Z-stacks to analyze three-dimensional distribution

  • Apply consistent exposure settings across experimental conditions

• Controls and validation:

  • Secondary antibody-only control to assess background

  • Preimmune serum control

  • Peptide competition assay

  • PP2B11 overexpression and knockout samples as positive/negative controls

Due to PP2B11's role in the SCF complex and its potential dynamic regulation under stress conditions, researchers should consider dual labeling with antibodies against known interaction partners or cellular markers for colocalization analysis.