FLS2 Antibody

What is FLS2 and why is it significant in plant immunity research?

FLS2 is a leucine-rich repeat receptor-like kinase that serves as a pattern recognition receptor in plants, particularly well-studied in Arabidopsis thaliana. It forms a dynamic complex with the co-receptor BAK1 and the receptor-like cytoplasmic kinase BIK1 to perceive the bacterial flagellar epitope flg22, triggering plant immune responses . FLS2 research is significant because it provides an excellent model for understanding plant innate immune signaling and receptor kinases in general, helping researchers unravel fundamental mechanisms of plant-pathogen interactions .

How does FLS2 signaling cascade function in plant defense responses?

The FLS2 signaling pathway operates through multiple coordinated mechanisms. Upon flg22 perception, FLS2 rapidly interacts with BAK1 to form an active receptor complex, initiating phosphorylation events through activating receptor-like cytoplasmic kinases such as BIK1 . In the pre-activation state, the heterotrimeric G protein XLG2 directly interacts with FLS2 and BIK1, functioning together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation . Following activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD . Additionally, phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics, enhancing flg22-induced FLS2 internalization and immune responses .

What are the key structural characteristics of FLS2 protein relevant for antibody development?

FLS2 is a single-pass membrane protein with a molecular weight of approximately 126 kDa (without propeptide) . Its structure includes an extracellular leucine-rich repeat domain that recognizes flagellin, a transmembrane domain, and an intracellular kinase domain. The immunogenic regions most suitable for antibody development typically come from unique peptide sequences specific to FLS2, such as those found in the Arabidopsis thaliana FLS2 sequence (UniProt: Q9FL28, TAIR: AT5G46330) . Understanding these structural characteristics is essential for developing specific antibodies that can effectively recognize FLS2 in different experimental contexts without cross-reactivity to related receptor kinases.

What criteria should researchers consider when selecting an FLS2 antibody?

When selecting an FLS2 antibody, researchers should consider multiple factors including specificity, sensitivity, application compatibility, and host species. For specificity, evaluate whether the antibody has been validated against knockout/knockdown controls to ensure it recognizes only FLS2 and not related receptor kinases. For sensitivity, review literature or validation data demonstrating detection limits relevant to your experimental system. The antibody should be validated for your specific application (Western blot, immunoprecipitation, immunofluorescence) . Additionally, consider the host species the antibody was raised in to avoid cross-reactivity in your experimental system, particularly if performing co-immunoprecipitation or immunohistochemistry with multiple antibodies.

How can researchers validate FLS2 antibody specificity in their experimental system?

A comprehensive validation approach includes multiple methods. First, perform Western blot analyses comparing wild-type plants with fls2 mutants or knockdown lines to confirm band disappearance in mutants. Second, pre-absorb the antibody with the immunizing peptide to demonstrate signal loss, confirming specificity. Third, conduct immunoprecipitation followed by mass spectrometry to verify that the antibody pulls down FLS2. Fourth, transiently express tagged FLS2 and perform parallel detection with both the FLS2 antibody and an antibody against the tag to confirm co-localization. Fifth, verify species reactivity if working with FLS2 from plants other than Arabidopsis thaliana, as antibodies like ABIN4966167 have predicted reactivity with FLS2 from Capsella rubella, Glycine max, Hordeum vulgare, Populus trichocarpa, and Vitis vinifera .

What controls are essential when using FLS2 antibodies in immunoblotting experiments?

Essential controls include: (1) Positive control: Arabidopsis thaliana wild-type tissue expressing FLS2; (2) Negative control: fls2 knockout/knockdown plant tissue; (3) Loading control: detection of a constitutively expressed protein (e.g., actin or tubulin) to ensure equal loading across samples; (4) Molecular weight marker: to confirm the expected 126 kDa size of FLS2 ; (5) Peptide competition: pre-incubation of the antibody with the immunizing peptide should abolish specific signals; (6) Secondary antibody-only control: to identify any non-specific binding from the secondary antibody. These controls collectively ensure that any detected signals are specifically attributable to FLS2 and not to experimental artifacts or cross-reactivity.

What is the optimal protocol for detecting FLS2 by Western blotting?

For optimal FLS2 detection by Western blotting, follow this protocol: (1) Extract total proteins from plant tissues using a buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% Triton X-100, and protease inhibitors; (2) For membrane proteins like FLS2, include a membrane protein extraction step; (3) Load 30-50μg of protein per lane on an 8% SDS-PAGE gel to efficiently separate the 126 kDa FLS2 protein; (4) Transfer to PVDF membrane (preferred over nitrocellulose for high molecular weight proteins) at low amperage overnight at 4°C; (5) Block with 5% non-fat dry milk in TBST for 1 hour; (6) Incubate with primary FLS2 antibody at 1:5000 dilution overnight at a 4°C; (7) Wash 3-5 times with TBST; (8) Incubate with HRP-conjugated secondary antibody for 1 hour; (9) Wash extensively and develop using enhanced chemiluminescence. This protocol maximizes specificity while minimizing background that can complicate interpretation of results with membrane proteins.

How can researchers optimize immunoprecipitation protocols for studying FLS2 protein interactions?

For effective immunoprecipitation of FLS2 and its interacting partners: (1) Harvest and flash-freeze plant tissue treated with flg22 at different time points (0, 5, 15, 30 min) to capture dynamic interactions; (2) Extract proteins using a mild lysis buffer (50mM HEPES pH 7.5, 150mM NaCl, 1% NP-40, 1mM EDTA) supplemented with phosphatase inhibitors and protease inhibitors; (3) Pre-clear lysates with protein A/G beads; (4) Immobilize FLS2 antibody on protein A/G beads using a chemical crosslinker to prevent antibody co-elution; (5) Incubate pre-cleared lysates with antibody-conjugated beads overnight at 4°C with gentle rotation; (6) Perform stringent washes (at least 5) with wash buffer containing reduced detergent; (7) Elute bound proteins with glycine buffer (pH 2.5) or by boiling in SDS sample buffer if the antibody is crosslinked; (8) Analyze by Western blot or mass spectrometry. For studying phosphorylation-dependent interactions, include phosphatase inhibitors throughout and consider phospho-specific antibodies to detect modification states of FLS2 or its partners .

What are the technical considerations for immunolocalization of FLS2 in plant tissues?

Immunolocalization of FLS2 requires attention to several technical aspects: (1) Fixation: Use 4% paraformaldehyde with 0.1% glutaraldehyde for 2-3 hours to preserve membrane proteins while maintaining tissue integrity; (2) Permeabilization: Optimize detergent concentration (0.1-0.3% Triton X-100) to allow antibody access without excessive extraction of membrane proteins; (3) Antigen retrieval: May be necessary with certain fixatives—test citrate buffer (pH 6.0) heating; (4) Blocking: Use 2-3% BSA with 0.05% Tween-20 to reduce non-specific binding; (5) Antibody concentration: Test dilutions ranging from 1:100 to 1:500 for primary antibody; (6) Incubation time: Extend to 24-48 hours at 4°C for thick plant tissues; (7) Washing: Perform extensive washes between steps; (8) Controls: Include peptide competition and fls2 mutant tissues; (9) Counterstaining: Use plasma membrane markers to confirm membrane localization. For co-localization studies with FLS2 partners such as BAK1 or BIK1, sequential or multi-color immunolabeling protocols should be optimized to minimize cross-reactivity .

How can FLS2 antibodies be utilized to study receptor oligomerization in response to flg22?

FLS2 receptor oligomerization can be studied using antibody-based approaches: (1) Co-immunoprecipitation combined with crosslinking: Apply membrane-impermeable crosslinkers of different arm lengths before cell lysis to capture transient interactions, then immunoprecipitate with FLS2 antibodies; (2) Proximity ligation assay (PLA): Use primary antibodies against FLS2 and potential interaction partners, followed by secondary antibodies conjugated to complementary oligonucleotides that generate fluorescent signals when proteins are in close proximity; (3) Blue native PAGE: Extract membrane proteins in non-denaturing conditions and separate native protein complexes before immunoblotting with FLS2 antibodies; (4) Förster resonance energy transfer (FRET) combined with immunostaining: Use fluorophore-conjugated FLS2 antibodies compatible with live-cell imaging to detect nanoscale proximity; (5) Electron microscopy with immunogold labeling: Visualize the spatial distribution and clustering of FLS2 at the ultrastructural level. These approaches can reveal how oligomerization states change over time after flg22 perception, as suggested by recent research showing that synthetic FLS2 receptor oligomers exhibit enhanced defense mechanisms in an oligomerization status-dependent manner .

What methods can detect phosphorylation-dependent changes in FLS2 using phospho-specific antibodies?

Phosphorylation-dependent changes in FLS2 can be detected using several complementary approaches: (1) Western blotting with phospho-specific antibodies: Generate antibodies that specifically recognize phosphorylated residues like Ser-938 and compare signal intensity in samples treated with flg22 versus controls; (2) Phos-tag SDS-PAGE: Use this technique to separate phosphorylated from non-phosphorylated FLS2 species, followed by immunoblotting with general FLS2 antibodies; (3) Mass spectrometry following immunoprecipitation: Enrich FLS2 using antibodies, then identify phosphorylation sites using MS/MS; (4) Multiplexed immunofluorescence: Combine phospho-specific and general FLS2 antibodies with different fluorophores to visualize the subcellular distribution of phosphorylated receptor pools; (5) Flow cytometry with phospho-FLS2 antibodies: Quantify phosphorylation events at the single-cell level; (6) Biolayer interferometry: Measure the binding kinetics between phospho-FLS2 and its interacting partners to determine how phosphorylation affects protein-protein interactions. These methods can help elucidate how phosphorylation impacts FLS2's spatiotemporal dynamics and lifetime at the plasma membrane, providing insight into the regulation of plant immune signaling .

How can researchers investigate the relationship between FLS2 and heterotrimeric G proteins using antibody-based approaches?

To investigate FLS2-G protein interactions, researchers can employ the following methods: (1) Sequential co-immunoprecipitation: First immunoprecipitate with anti-FLS2 antibodies, then perform a second immunoprecipitation with antibodies against G protein components (XLG2, AGB1, AGG1/2) to identify complex composition; (2) Proximity-dependent biotin labeling: Express BirA-tagged FLS2 in planta, purify biotinylated proteins with streptavidin, and detect G proteins using specific antibodies; (3) Immunofluorescence co-localization with super-resolution microscopy: Use differentially labeled antibodies against FLS2 and G proteins to determine spatial relationships at the nanoscale; (4) Protein fragment complementation assays: Combine with immunodetection to validate interactions observed in cell-free systems; (5) Chromatin immunoprecipitation (ChIP): If studying transcriptional regulation, use antibodies against both FLS2 signaling components and G proteins to identify co-regulated genomic regions; (6) Time-course studies: Analyze samples at different time points after flg22 treatment to track the dissociation of XLG2 from AGB1 and subsequent phosphorylation by BIK1, which are key events in FLS2-mediated immune signaling .

What are common challenges in FLS2 antibody-based experiments and how can they be addressed?

How can researchers distinguish between specific and non-specific signals when using FLS2 antibodies?

To distinguish specific from non-specific signals: (1) Genetic controls: Always include fls2 mutant or knockout samples where the specific signal should be absent; (2) Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide—specific signals should disappear while non-specific binding remains; (3) Gradient of protein loading: Specific signals should change proportionally with protein amount while non-specific signals may not follow this pattern; (4) Signal size verification: Confirm that the detected band corresponds to the expected 126 kDa size of FLS2 ; (5) Multiple antibodies approach: If available, use different antibodies raised against distinct epitopes of FLS2—specific signals should be consistent across antibodies; (6) Treatment-induced changes: Verify that signals change as expected with flg22 treatment, which should alter FLS2 phosphorylation and complex formation ; (7) Fractionation experiments: Confirm that FLS2 signals are enriched in membrane fractions consistent with its known localization.

How should researchers interpret contradictory results from different antibody-based methods when studying FLS2?

When facing contradictory results: (1) Evaluate method-specific limitations: Each technique has inherent biases—for instance, Western blotting may not capture conformational epitopes present in native conditions; (2) Consider epitope accessibility: The epitope recognized by the antibody may be masked in certain protein conformations or complexes; (3) Examine buffer conditions: Different buffers can affect protein conformation and antibody binding; (4) Assess temporal dynamics: FLS2 undergoes rapid changes following flg22 perception, so timing differences between experiments could explain discrepancies; (5) Quantify results: Use quantitative rather than qualitative assessments when possible, including statistical analyses of multiple replicates; (6) Validate with complementary approaches: Confirm findings using non-antibody methods such as mass spectrometry or genetic approaches; (7) Examine experimental context: Consider whether differences in plant growth conditions, tissue types, or flg22 concentrations could explain contradictory results. When interpreting such contradictions, prioritize results from methods with the most robust controls and consider developing a model that accommodates seemingly contradictory data by accounting for dynamic, context-dependent regulation of FLS2 .

How can FLS2 antibodies contribute to studying spatiotemporal dynamics of immune receptor complexes?

FLS2 antibodies can provide valuable insights into spatiotemporal dynamics through several advanced approaches: (1) Single-molecule tracking using fluorescently labeled FLS2 antibody fragments (Fab or scFv) to visualize receptor diffusion and clustering in live cells with minimal perturbation; (2) Super-resolution microscopy combined with immunolabeling to visualize nanoclusters and their rearrangement following immune stimulation; (3) Optogenetic tools coupled with immunodetection to track rapid conformational changes in the receptor complex; (4) Microfluidic systems that allow precise temporal control of flg22 application while performing real-time immunofluorescence; (5) Correlative light and electron microscopy (CLEM) with immunogold labeling to connect functional observations with ultrastructural context. These approaches can help elucidate how phosphorylation of specific residues like Ser-938 enhances flg22-induced FLS2 internalization and immune responses by partitioning FLS2 into functional nanodomains, as recent research has demonstrated .

What role might FLS2 antibodies play in investigating the relationship between receptor oligomerization and immune signaling?

FLS2 antibodies can significantly advance understanding of the relationship between receptor oligomerization and signaling by: (1) Capturing different oligomeric states through carefully timed crosslinking and immunoprecipitation after flg22 treatment; (2) Identifying oligomerization-specific post-translational modifications through immunoprecipitation followed by mass spectrometry; (3) Determining the composition of different oligomeric complexes at various time points after immune activation; (4) Visualizing the formation and dissolution of signaling platforms through multi-color super-resolution imaging; (5) Assessing how mutations affecting oligomerization impact downstream signaling through phospho-specific antibodies against pathway components. These approaches can build upon recent findings showing that synthetic FLS2 receptor oligomers exhibit enhanced defense mechanisms depending on their oligomerization status, with dimerization significantly enhancing immune responses while tetrameric versions impair receptor endocytosis, disrupting timely turnover and weakening sustained immune signaling .

How can integrative approaches combining antibody-based detection with other techniques enhance FLS2 research?

Integrative approaches offer powerful insights by overcoming limitations of individual methods: (1) Combine CRISPR-Cas9 genome editing of endogenous FLS2 with antibody-based tracking to study receptor variants in their native context; (2) Pair single-cell transcriptomics with immunofluorescence to correlate FLS2 protein levels with gene expression patterns in individual cells; (3) Integrate structural biology approaches (X-ray crystallography, cryo-EM) with epitope mapping using antibody fragments to link structure to function; (4) Combine biosensors for secondary messengers (ROS, Ca²⁺) with immunodetection to connect receptor activation to downstream signaling events with temporal precision; (5) Use systems biology approaches to model receptor dynamics based on quantitative antibody-derived data across multiple timescales; (6) Implement machine learning algorithms to analyze complex patterns in high-content screening data generated using FLS2 antibodies. These integrative approaches can help resolve contradictions in the literature and provide a more comprehensive understanding of how FLS2 and its partners like heterotrimeric G proteins coordinate to regulate plant immune responses through both pre-activation and post-activation mechanisms .