PBL11 Antibody

What is PBL11 and what is its biological significance?

PBL11 (UniProt ID: P43293) belongs to the PBS1-Like (PBL) family of receptor-like cytoplasmic kinases in Arabidopsis thaliana. Based on homology to other PBL proteins, it likely functions in plant immune signaling pathways, potentially connecting pattern recognition receptors to downstream immune responses. While specific information about PBL11 is limited in the current literature, related PBL family members such as PBL27 are known to function in PAMP-triggered immunity by linking cell surface receptors to intracellular signaling cascades .

How does PBL11 compare structurally and functionally to other PBL family proteins?

PBL11 likely shares structural features with other PBL family proteins, including a kinase domain that mediates phosphorylation of downstream targets. The most well-characterized family member, PBL27, is known to function downstream of the chitin receptor CERK1 and interacts with MAPKKK5 at the plasma membrane to connect chitin recognition to MAPK cascade activation . PBL11 may have similar or distinct roles in immune signaling. Sequence analysis would reveal conserved domains and potential phosphorylation sites that could provide insights into its specific function within plant immunity pathways.

What types of PBL11 antibodies are currently available for research?

Based on the catalog data, a rabbit polyclonal PBL11 antibody (catalog number CSB-PA331789XA01DOA) specific for Arabidopsis thaliana is commercially available . This antibody targets the protein encoded by UniProt ID P43293 and is supplied in two size options (2ml/0.1ml). While specific application validations are not detailed in the provided information, polyclonal antibodies typically work across multiple applications including Western blotting, immunoprecipitation, and immunofluorescence microscopy.

What are the optimal conditions for using PBL11 antibody in Western blot applications?

While specific optimization data for the PBL11 antibody is not provided, researchers should consider the following protocol based on similar plant protein antibodies:

• Sample preparation: Extract total protein from plant tissue using a buffer containing protease and phosphatase inhibitors

• Protein separation: Load 20-50 μg protein per lane on SDS-PAGE

• Transfer: Use PVDF membrane with standard wet transfer (100V for 60-90 minutes)

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

• Primary antibody: Start with 1:500-1:2000 dilution (based on similar antibodies like PATL1)

• Incubation: Overnight at 4°C with gentle agitation

• Washing: 3-5 washes with TBST, 5-10 minutes each

• Secondary antibody: Anti-rabbit HRP conjugate at 1:5000-1:10000

• Detection: ECL substrate with appropriate exposure time

Researchers should validate and optimize these conditions for their specific experimental system.

How can I design an immunoprecipitation protocol using PBL11 antibody?

Based on protocols for similar antibodies like PATL1 , an effective immunoprecipitation protocol would include:

• Sample preparation:

  • Homogenize 1-2g of plant tissue in extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, protease and phosphatase inhibitors)

  • Clarify by centrifugation at 12,000g for 15 minutes at 4°C

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 0.5-4.0 μg of PBL11 antibody per 1.0-3.0 mg of total protein

  • Incubate 4-16 hours at 4°C with rotation

  • Add fresh Protein A/G beads and incubate 2-4 hours at 4°C

  • Wash 4-5 times with extraction buffer

  • Elute with SDS sample buffer at 95°C for 5 minutes

• Analysis:

  • Run SDS-PAGE followed by Western blotting or mass spectrometry

What strategies can be used to verify PBL11 antibody specificity?

To confirm PBL11 antibody specificity, implement multiple validation approaches:

• Genetic controls:

  • Compare signal between wild-type and pbl11 knockout/knockdown plants

  • Include overexpression lines as positive controls

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide/recombinant protein

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific signals should disappear with peptide competition

• Mass spectrometry validation:

  • Perform immunoprecipitation with PBL11 antibody

  • Analyze by LC-MS/MS to confirm identity of captured proteins

  • Check for presence of PBL11-specific peptides

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of closely related PBL family members

  • Evaluate recognition patterns across protein family

How can PBL11 antibody be used to study protein-protein interactions in immune signaling pathways?

Multiple approaches can be employed to investigate PBL11 interaction networks:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate PBL11 using the specific antibody

  • Analyze co-precipitating proteins by Western blot or mass spectrometry

  • Based on the PBL27 model, consider testing interactions with CERK1 and components of MAPK cascades

  • Perform reciprocal Co-IPs to confirm interactions

• Proximity-dependent labeling:

  • Combine immunoprecipitation with crosslinking approaches

  • Use chemical crosslinkers to stabilize transient interactions

  • Similar to techniques used for studying PBL27-MAPKKK5 interactions at the plasma membrane

• Stimulus-dependent interaction analysis:

  • Compare protein complexes before and after PAMP treatment

  • Track dynamic association/dissociation events

  • Similar to the observed chitin-induced dissociation of PBL27-MAPKKK5 complex

• Subcellular co-localization:

  • Use immunofluorescence microscopy with PBL11 antibody and antibodies against potential interactors

  • Analyze spatial proximity at different timepoints after immune stimulation

What approaches can be used to analyze PBL11 phosphorylation status during immune responses?

To investigate PBL11 phosphorylation dynamics:

• Phos-tag gel electrophoresis:

  • Separate phosphorylated and non-phosphorylated forms

  • Detect with PBL11 antibody by Western blot

  • Compare mobility shifts after PAMP treatments

  • Include phosphatase-treated controls

• Targeted phosphosite analysis:

  • Immunoprecipitate PBL11 after various treatments

  • Perform phospho-peptide enrichment

  • Identify and quantify phosphorylation sites by mass spectrometry

  • Compare to known phosphosites in PBL27 or other family members

• In vitro kinase assays:

  • Express recombinant PBL11

  • Test phosphorylation by candidate upstream kinases (e.g., CERK1)

  • Analyze autophosphorylation capacity

  • Identify potential downstream substrates

• Phosphospecific antibodies:

  • If available, use phospho-specific antibodies targeting PBL11 phosphosites

  • Monitor phosphorylation dynamics during immune responses

How can PBL11 antibody contribute to understanding spatial and temporal dynamics of immune signaling?

For spatial-temporal analysis of PBL11 function:

• Subcellular fractionation:

  • Separate membrane, cytosolic, and nuclear fractions

  • Track PBL11 localization before and after immune stimulation

  • Similar to approaches used for PBL27 that demonstrated plasma membrane localization

• Time-course experiments:

  • Collect samples at short intervals after PAMP treatment

  • Monitor changes in PBL11 phosphorylation, localization, and interaction partners

  • Create temporal maps of signaling events

  • Compare with known dynamics of PBL27-MAPKKK5 disassociation after chitin treatment

• Tissue-specific analysis:

  • Use immunohistochemistry to analyze PBL11 expression across different tissues

  • Compare expression patterns with known immune receptors

  • Correlate with sites of pathogen entry or immune response

• Single-cell resolution studies:

  • Combine with cell-type specific markers

  • Analyze cell-to-cell variation in PBL11 levels and localization

Why might I observe multiple bands or inconsistent results with PBL11 antibody in Western blots?

Multiple factors could contribute to unexpected Western blot results:

• Post-translational modifications:

  • Phosphorylation can cause mobility shifts

  • Other modifications (ubiquitination, sumoylation) may result in higher molecular weight bands

  • Compare with samples treated with phosphatases or deubiquitinating enzymes

• Sample preparation issues:

  • Protein degradation during extraction (add more protease inhibitors)

  • Incomplete denaturation (increase SDS concentration and heating time)

  • Sample overloading (dilute samples and re-run)

  • Membrane blocking inefficiency (try alternative blocking agents)

• Antibody specificity considerations:

  • Cross-reactivity with related PBL family proteins

  • Non-specific binding (increase antibody dilution, optimize washing)

  • Batch-to-batch variation (compare lot numbers)

• Detection system problems:

  • Secondary antibody cross-reactivity

  • Over-development of signal (reduce exposure time)

  • Insufficient signal (increase protein loading or antibody concentration)

How can I optimize immunoprecipitation of PBL11 for detecting transient interactions?

For capturing dynamic or transient interactions:

• Chemical crosslinking approaches:

  • Use membrane-permeable crosslinkers like DSP or formaldehyde

  • Apply brief crosslinking (1-5 minutes) immediately after stimulation

  • Stabilize complexes before cell lysis

  • Similar to approaches that captured PBL27-MAPKKK5 interactions

• Modified buffer conditions:

  • Use gentler detergents (digitonin, CHAPS) instead of stronger ones

  • Include stabilizing agents (10% glycerol, specific ions)

  • Maintain physiological pH and salt concentrations

  • Add phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Rapid sample processing:

  • Minimize time between stimulation and lysis

  • Process samples at 4°C throughout

  • Consider using GFP-tagged proteins and GFP-Trap for faster pulldowns

• Sequential elution strategies:

  • Use different elution conditions to identify interaction strength

  • Compare salt-sensitive versus detergent-sensitive interactions

What strategies can address non-specific binding issues when using PBL11 antibody?

To minimize non-specific binding:

• Blocking optimization:

  • Test alternative blocking agents (BSA, casein, commercial blockers)

  • Extend blocking time or increase blocking agent concentration

  • Add blocking agent to antibody dilution buffer

• Antibody conditions:

  • Further dilute primary antibody

  • Reduce incubation time or temperature

  • Pre-absorb antibody with plant lysate from pbl11 knockout plants

• Washing optimization:

  • Increase number of washes

  • Use higher detergent concentration (0.1-0.3% Tween-20)

  • Add low concentrations of salt (150-300mM NaCl) to wash buffer

• Sample preparation:

  • Pre-clear lysates with beads before adding antibody

  • Filter lysates to remove aggregates

  • Ultracentrifuge to remove membrane fragments and vesicles

How does PBL11 function compare with PBL27 in immune signaling pathways?

Based on what is known about PBL27, we can propose comparative studies:

• Receptor specificity:

  • PBL27 functions downstream of the chitin receptor CERK1

  • PBL11 may associate with different pattern recognition receptors

  • Test receptor associations through co-immunoprecipitation experiments

• Downstream targets:

  • PBL27 interacts with and phosphorylates MAPKKK5

  • PBL11 may target different MAPKKKs or other signaling components

  • Compare substrate specificity through kinase assays

• Activation dynamics:

  • PBL27-MAPKKK5 interaction is disrupted after chitin treatment but not flg22

  • PBL11 may show different PAMP-specific response patterns

  • Compare interaction dynamics after various PAMP treatments

• Signaling outcomes:

  • Analyze defense gene expression in pbl11 versus pbl27 mutants

  • Compare contributions to different branches of immunity

  • Assess potential redundancy through double mutant analysis

How can multiparametric analysis with PBL11 antibody reveal complex immune signaling networks?

To investigate complex signaling networks:

• Multiplex immunodetection:

  • Simultaneously detect PBL11 with other signaling components

  • Use different fluorescent labels for co-localization studies

  • Track multiple proteins during immune responses

• Sequential immunoprecipitation:

  • First IP with PBL11 antibody

  • Elute complexes and perform second IP with antibodies against other components

  • Identify proteins that participate in multiple complexes

  • Compare with known PBL27-containing complexes

• Integration with phosphoproteomics:

  • Compare phosphoproteome changes in wild-type versus pbl11 mutants

  • Identify potential PBL11-dependent phosphorylation events

  • Map kinase-substrate networks

• Correlation analysis:

  • Track PBL11 activation alongside MAPK activation

  • Create temporal maps of signaling events

  • Similar to analysis of PBL27-MAPKKK5-MAPK pathway dynamics

How can systems biology approaches with PBL11 antibody advance understanding of plant immunity?

For systems-level investigations:

• Integrative "omics" approaches:

  • Combine PBL11 antibody-based proteomics with transcriptomics

  • Correlate protein complex dynamics with gene expression changes

  • Map temporal sequence of events in immune activation

• Mathematical modeling:

  • Develop quantitative models of signaling dynamics

  • Compare with existing models of PBL27-MAPK pathway

  • Predict system behaviors under different conditions

• Network analysis:

  • Position PBL11 within broader immune signaling networks

  • Compare with networks involving PBL27 and other PBL family members

  • Identify shared and unique components and connections

• Cross-species comparative analysis:

  • Use antibodies against PBL11 orthologs in other plant species

  • Compare conservation of signaling mechanisms

  • Relate to evolutionary aspects of plant immunity

How might PBL11 antibody contribute to understanding cross-talk between different immune pathways?

PBL11 antibody can be leveraged to investigate pathway cross-talk:

• Dual PAMP stimulation experiments:

  • Apply multiple PAMPs in different sequences

  • Track PBL11 phosphorylation and complex formation

  • Compare with PBL27 responses that show specificity for chitin but not flg22

  • Analyze how one pathway affects signaling through another

• Hormone-immune interactions:

  • Treat plants with immune hormones (salicylic acid, jasmonic acid)

  • Monitor effects on PBL11 status and interactions

  • Investigate how hormonal priming affects PBL11-mediated signaling

• Abiotic-biotic stress cross-talk:

  • Apply abiotic stresses before PAMP treatment

  • Analyze changes in PBL11 complex formation and function

  • Determine how environmental factors modulate immune responses

• Multi-receptor integration:

  • Compare PBL11 activation downstream of different PRRs

  • Investigate potential convergence or divergence in signaling

  • Similar to the approach that revealed PBL27 specificity in chitin but not flg22 responses

What role might PBL11 play in broad-spectrum versus specific immunity?

To investigate PBL11's contribution to immunity specificity:

• Pathogen challenge experiments:

  • Compare susceptibility of pbl11 mutants to diverse pathogens

  • Analyze which defense responses require PBL11 function

  • Contrast with known PBL27 contributions to chitin-triggered immunity

• PAMP responsiveness spectrum:

  • Test PBL11 activation by diverse PAMPs (bacterial, fungal, oomycete)

  • Compare with the chitin-specific response of PBL27

  • Determine if PBL11 contributes to broad-spectrum or specific immune branches

• Defense output analysis:

  • Use PBL11 antibody to correlate protein activation with defense markers

  • Compare profiles of antimicrobial compounds in wild-type versus mutants

  • Analyze transcriptional reprogramming dependencies

• Evolution and conservation:

  • Compare PBL11 orthologs across plant species

  • Relate sequence conservation to functional conservation

  • Investigate emergence of specificity in the PBL family

How can emerging technologies enhance PBL11 antibody applications in research?

Novel technological approaches include:

• Proximity labeling techniques:

  • Combine PBL11 antibody with TurboID or APEX2-based proximity labeling

  • Map protein neighborhoods around PBL11 in vivo

  • Identify transient or weak interactors missed by traditional IP

• Super-resolution microscopy:

  • Use PBL11 antibody with STORM or PALM techniques

  • Visualize nanoscale organization of signaling complexes

  • Track dynamic reorganization during immune responses

• Single-cell proteomics:

  • Apply PBL11 antibody in single-cell resolution studies

  • Analyze cell-to-cell variation in immune responses

  • Identify specialized cell populations in immune signaling

• CRISPR-based approaches:

  • Generate endogenously tagged PBL11 for antibody-free detection

  • Create phospho-mimetic or phospho-dead mutants

  • Test functional hypotheses derived from antibody-based studies