RLP51 Antibody

What is RLP51 and why is it significant in plant immunity research?

RLP51 (also known as AtRLP51, PDO1, or SNC2) is a receptor-like protein in Arabidopsis thaliana that plays a critical role in plant innate immunity. It belongs to a family of surface immune receptors that detect pathogen-associated molecular patterns.

RLP51 has been identified as involved in basal resistance against bacterial pathogens (Pseudomonas syringae pv. phaseolicola), making it an important subject for plant-pathogen interaction studies . Notably, RLP51 is the underlying gene of the snc2-1D locus (suppressor of npr1, constitutive 2-1D), a semidominant gain-of-function mutant with dwarf morphology and constitutively activated defense responses, including elevated salicylic acid and PATHOGENESIS-RELATED (PR) gene levels .

What applications are recommended for RLP51 antibodies?

RLP51 antibodies have been validated for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of RLP51 in plant extracts

• Western Blot (WB): For identification of RLP51 protein in denatured samples

The antibody specificity should be confirmed through proper controls, as multiple commercial vendors indicate their antibodies are specifically validated for these two applications. Research protocols typically use either application to monitor RLP51 expression in various plant immunity studies.

How should RLP51 antibodies be stored and handled for optimal performance?

For maintaining antibody integrity and performance:

• Storage temperature: Store at -20°C or -80°C immediately upon receipt

• Avoid repeated freeze-thaw cycles: This can degrade antibody quality and reduce sensitivity

• Buffer composition: Typically preserved in 0.03% Proclin 300 with 50% Glycerol in 0.01M PBS (pH 7.4)

• Working dilutions: Prepare fresh dilutions for each experiment rather than storing diluted antibody

Storage conditions significantly impact antibody binding efficiency, with studies showing that improper storage can lead to up to 50% reduction in signal intensity in subsequent experiments.

How does RLP51 interact with other immune components in the plant signaling network?

RLP51 operates within a complex network of plant immune receptors. Research methodological approaches should consider:

• Co-immunoprecipitation experiments: For studying RLP51 interactions with regulatory receptor-like kinases such as BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR)

• Phosphorylation analysis: As demonstrated with related RLPs, phosphorylation status can be critical for signaling activity

• Protein complex formation: RLP51 likely forms protein complexes similar to other RLPs like RLP30, which physically interacts with SOBIR1 in a ligand-independent manner

When designing experiments to study RLP51 interactions, researchers should consider using both anti-RLP51 antibodies and antibodies against potential interacting partners, along with appropriate negative controls to validate specificity of interactions.

What are the key considerations for detecting RLP51 in Western blot experiments?

When performing Western blot analysis with RLP51 antibodies:

• Sample preparation:

  • Use fresh plant material whenever possible

  • Extract proteins using buffer containing 50mM PIPES, 5mM EGTA, 5mM MgSO₄ (pH 7.0)

  • Include protease inhibitors to prevent degradation

• Gel selection and transfer conditions:

  • 8-10% SDS-PAGE gels are typically suitable

  • Transfer to PVDF membranes at 100V for 60-90 minutes

• Antibody dilution optimization:

  • Typically used at 1:1000 to 1:2000 dilution

  • Incubate overnight at 4°C for optimal sensitivity

• Controls:

  • Include protein from rlp51 knockout plants as negative control

  • Use recombinant RLP51 protein (when available) as positive control

  • Consider using antigen competition to confirm specificity

Research has shown that optimization of blocking conditions (3-5% BSA rather than milk) can significantly improve detection specificity when working with plant receptor proteins.

How can RLP51 antibodies be validated for cross-reactivity and specificity?

Robust validation of RLP51 antibodies should include:

• Testing against knockout mutants: Use tissue from rlp51 mutant plants as negative controls

• Antigen competition assays: Pre-incubating the antibody with purified antigen should abolish specific signals

• Cross-reactivity assessment: Test against closely related RLPs (particularly RLP52) to ensure specificity

• Multiple detection methods: Confirm results using both immunoblotting and immunolocalization techniques

A comparative analysis of antibody specificity should be conducted when using antibodies from different commercial sources, as it has been shown that different polyclonal antibodies raised against the same antigen can have different epitope preferences.

What techniques are most effective for studying RLP51 localization in plant cells?

For subcellular localization studies of RLP51:

• Immunofluorescence microscopy:

  • Fix tissues with 3.7% formaldehyde in PBS

  • Permeabilize cell walls with 2% driselase

  • Use 1:75 to 1:100 dilution of primary RLP51 antibody

  • Visualize with fluorescently labeled secondary antibodies (1:100 dilution)

  • Include DAPI staining for nuclear reference

• Subcellular fractionation followed by immunoblotting:

  • Separate membrane, cytosolic, and nuclear fractions

  • Use marker proteins for each fraction (e.g., H⁺-ATPase for plasma membrane)

  • Probe with RLP51 antibodies to determine subcellular distribution

• Controls for localization specificity:

  • Include peptide competition controls

  • Use rlp51 mutant tissues as negative controls

The resolution of confocal microscopy (approximately 200nm) is sufficient for distinguishing membrane localization from cytoplasmic distribution of RLP51.

How does RLP51 expression change during pathogen infection?

RLP51 expression dynamics can be monitored using:

• Time-course analysis:

  • Collect samples at multiple timepoints after pathogen challenge

  • Compare expression levels using Western blot with RLP51 antibodies

  • Quantify band intensity relative to appropriate loading controls

• Tissue-specific expression patterns:

  • Compare expression in different plant tissues (roots, leaves, stems)

  • Correlate with tissue-specific pathogen susceptibility

Research has shown that many RLPs, including RLP51, are upregulated following pathogen treatment, with expression changes detected as early as 3-6 hours post-infection .

How can RLP51 antibodies be used to study plant immune receptor complexes?

To investigate RLP51's role in immune receptor complexes:

• Co-immunoprecipitation (Co-IP) approaches:

  • Use anti-RLP51 antibodies conjugated to agarose or magnetic beads

  • Lyse plant tissues in mild, non-denaturing conditions

  • Perform Western blot analysis of precipitated material to identify interacting partners

  • Confirm interactions using reverse Co-IP

• Blue Native-PAGE analysis:

  • Solubilize membranes using mild detergents

  • Separate native protein complexes

  • Perform Western blot with anti-RLP51 antibodies

  • Identify complex sizes and components

Studies with related RLPs have demonstrated that these receptors form multiprotein complexes with regulatory receptor-like kinases. For instance, RLP30 physically interacts with SOBIR1 in a ligand-independent manner, a pattern that might be conserved for RLP51 .

What considerations are important when using RLP51 antibodies in comparative genomics research?

When using RLP51 antibodies across different plant species:

• Epitope conservation analysis:

  • Compare the amino acid sequence of RLP51 between species

  • Focus on regions containing the immunizing epitope

  • Perform sequence alignments to predict cross-reactivity potential

• Validation in each species:

  • Always validate antibody specificity in each new species

  • Use tissue from corresponding mutants when available

  • Consider Western blot with recombinant protein standards

• Increased antibody concentration:

  • When working with less conserved RLP51 orthologs, higher antibody concentrations may be required

  • Typically 1.5-2× the concentration used for Arabidopsis

Research has shown that RLP family proteins show approximately 60-75% sequence conservation between Arabidopsis and other Brassicaceae species, but much lower conservation with more distant plant families .

What strategies can resolve weak or absent RLP51 signal in Western blots?

When facing detection challenges:

Most membrane proteins like RLPs require specific solubilization conditions; standard RIPA buffers may not efficiently extract RLP51 from plant membranes.

How can researchers distinguish between specific and non-specific binding of RLP51 antibodies?

To ensure signal specificity:

• Essential controls:

  • Knockout or knockdown plant tissues

  • Secondary antibody-only controls

  • Antigen competition/neutralization tests

• Signal validation approaches:

  • Compare results from multiple antibodies targeting different RLP51 epitopes

  • Correlate protein detection with transcript levels

  • Compare band patterns with expected molecular weight

• Advanced validation:

  • Immunoprecipitation followed by mass spectrometry

  • Correlation with fluorescently tagged RLP51 expression patterns

For confident interpretation, specific RLP51 signals should be absent in knockout mutants, reduced in knockdown lines, and blocked by pre-incubation with the immunizing peptide .

How can phospho-specific antibodies be developed to study RLP51 activation state?

Development of phospho-specific RLP51 antibodies:

• Phosphorylation site identification:

  • Use mass spectrometry to identify in vivo phosphorylation sites

  • Focus on the cytoplasmic domain and potential regulatory sites

  • Compare with known phosphorylation sites in related RLPs

• Phosphopeptide antibody generation:

  • Design phosphopeptide antigens covering identified sites

  • Raise antibodies in rabbits for polyclonal production

  • Screen for phospho-specific recognition

  • Validate using phosphatase-treated samples

• Application considerations:

  • Use phosphatase inhibitors during sample preparation

  • Include phosphorylated and non-phosphorylated peptide controls

  • Consider using Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated forms

Similar approaches with related immune receptors have shown that phospho-specific antibodies can effectively monitor receptor activation status in response to pathogen treatment .

How does RLP51 compare to other RLPs in plant immunity studies?

Comparative analysis of RLP family members:

• Functional comparison:

  • RLP30, RLP51, and RLP52 are all involved in basal resistance to pathogens

  • RLP30 specifically recognizes the SCLEROTINIA CULTURE FILTRATE ELICITOR1 (SCFE1)

  • RLP51 (SNC2) mediates constitutive defense responses when mutated

  • RLP52 contributes to resistance against the fungus Erysiphe cichoracearum

• Structural considerations for antibody design:

  • RLPs contain conserved leucine-rich repeat (LRR) domains

  • Most specific epitopes are found in the variable N-terminal regions

  • C-terminal regions often contain conserved signaling motifs

• Experimental approach differences:

  • Unlike some RLPs, RLP51 appears in both pathogen-responsive clusters and developmental clusters in expression studies

  • RLP51 belongs to the "PUTATIVE DEVELOPMENTAL ORTHOLOG" (PDO) group, yet functions in pathogen defense

Understanding these relationships is critical when designing experiments to study RLP51-specific functions versus shared RLP family functions.

What novel techniques are emerging for studying RLP51 dynamics in living tissues?

Cutting-edge approaches for RLP51 research:

• CRISPR-tagged endogenous RLP51:

  • Generate epitope-tagged RLP51 at its native locus

  • Use antibodies against the tag for enhanced specificity

  • Allows monitoring of RLP51 without overexpression artifacts

• Single-molecule tracking approaches:

  • Combine antibody fragment labeling with super-resolution microscopy

  • Track RLP51 movement and clustering during immune activation

  • Correlate with co-receptor dynamics

• Proximity labeling techniques:

  • Fuse RLP51 to promiscuous biotin ligases (BioID/TurboID)

  • Identify proximal proteins using streptavidin pulldown

  • Map the dynamic RLP51 interactome during immune responses

These emerging techniques provide spatial and temporal resolution exceeding traditional antibody-based methods, allowing researchers to address fundamental questions about RLP51 dynamics in living tissues.

How should researchers approach contradictory results when using different RLP51 antibodies?

When facing contradictory antibody results:

• Systematic validation approach:

  • Map the exact epitopes recognized by different antibodies

  • Test each antibody against recombinant RLP51 fragments

  • Validate using genetic controls (knockouts, overexpression)

• Technical considerations:

  • Different fixation methods may preserve different epitopes

  • Native versus denatured conditions affect epitope accessibility

  • Post-translational modifications may mask epitopes

• Reconciliation strategies:

  • Use multiple antibodies targeting different regions

  • Correlate antibody results with orthogonal techniques (e.g., GFP-fusion proteins)

  • Consider reporting all results with detailed methodological differences

Research has demonstrated that antibody discrepancies often reflect biological reality rather than technical artifacts, such as detecting different protein isoforms, conformational states, or post-translational modifications .