FD2 Antibody

What is FD2 and why is it significant in plant research?

Ferredoxin 2 (Fd2) is a major leaf ferredoxin that functions as a key electron distributor in plastids, contributing significantly to redox regulation and antioxidant defense mechanisms in plants. The protein has gained substantial research interest because it plays a crucial regulatory role in plant innate immunity, particularly in Arabidopsis thaliana. Studies have demonstrated that Fd2 expression is suppressed during Pseudomonas syringae pv. tomato (Pst) DC3000 infection, and knockout mutants (Fd2-KO) display increased susceptibility to both Pst DC3000 and Golovinomyces cichoracearum pathogens . These findings highlight Fd2's importance as a molecular link between photosynthetic processes and defense responses, making it an excellent target for studying how plants balance energy production with immune responses. Understanding Fd2 function has implications for developing strategies to enhance crop resistance to pathogens while maintaining photosynthetic efficiency.

What types of FD2 antibodies are currently available for plant research?

Based on the available search results, researchers can utilize polyclonal Anti-Fd2 antibodies for their investigations. These antibodies are typically raised in rabbit hosts and show confirmed reactivity with Arabidopsis thaliana, Synechocystis PCC 6803, and Zea mays Fd2 proteins . Commercial antibodies are generally supplied as total IgG that has been purified using Protein A chromatography and formulated in PBS with 50% glycerol for stability . The immunogen used to generate these antibodies is typically full-length, recombinant maize Fd2 with cleaved tags, such as the one corresponding to UniProt accession P16972 . These polyclonal antibodies offer broad cross-species reactivity, with predicted recognition of Fd2 from additional plant species including Brachypodium distachyon, Oryza sativa, Setaria italica, and Sorghum bicolor . This versatility makes these antibodies valuable tools for comparative studies across different plant model systems.

What are the recommended applications and protocols for using FD2 antibodies?

FD2 antibodies have been validated for several research applications with specific recommended protocols for optimal results:

Western Blot Analysis:

• Recommended dilution range: 1:1000 to 1:5000

• Sample preparation: Extract plant tissue with 2x SDS-sample buffer containing 2-mercaptoethanol

• Recommended loading: 10 μg total leaf extract per well

• Separation: 10% SDS-PAGE followed by 1-hour transfer to PVDF membrane

• Blocking: 3% skim milk in TBS-T for 1 hour at room temperature with agitation

• Primary antibody incubation: Anti-Fd2 at 1:1000 dilution in TBS-T for 1 hour at room temperature

• Washing: 4 times for 10 minutes each in TBS-T

• Secondary antibody: Anti-rabbit IgG-HRP conjugate at 1:10,000 dilution for 1 hour

• Detection: Chemiluminescent substrate following manufacturer's recommendations

Immunoprecipitation:

• Extraction buffer: 150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl (pH 8.0)

• Denaturation: 4X SDS buffer at 95°C for 5 minutes before electrophoresis

ELISA:

• Antibodies have been validated for ELISA applications, making them suitable for quantitative analysis of Fd2 in plant extracts

When working with these antibodies, researchers should note that the expected molecular weight of Fd2 is approximately 15 kDa, and proper storage at -20°C with aliquoting to avoid freeze-thaw cycles is recommended for maintaining antibody performance .

What is the molecular weight and structure of the FD2 protein, and how does this inform antibody selection?

The Ferredoxin 2 (Fd2) protein has an expected molecular weight of approximately 15 kDa as detected in Western blot analysis . While detailed structural information is not provided in the search results, Fd2 belongs to the ferredoxin family, which typically contains iron-sulfur clusters essential for electron transfer functions. The protein is localized primarily in chloroplasts, with the interesting distinction that it can also be found in stromules (stroma-filled tubular extensions) that extend from chloroplasts .

This localization pattern has important implications for antibody selection and experimental design:

• For immunofluorescence studies targeting Fd2, researchers should select antibodies that maintain reactivity under fixation conditions required for visualizing both chloroplast bodies and stromule extensions.

• When designing experiments to study Fd2's distinctive stromule localization, consider using dual-labeling approaches with chloroplast markers to clearly distinguish between chloroplast body and stromule-associated Fd2.

• For biochemical studies, researchers should ensure complete extraction of both soluble chloroplast proteins and those associated with membrane extensions, potentially requiring optimization of extraction buffers beyond standard protocols.

• When validating knockout or knockdown lines, antibodies should be tested against expected molecular weight markers, with careful attention to potential cross-reactivity with other ferredoxin isoforms that may have similar molecular weights.

Understanding these structural and localization characteristics helps researchers select the most appropriate antibody format and experimental conditions for their specific Fd2 studies.

How does FD2 regulate plant innate immunity, and what methodological approaches can reveal these mechanisms?

Fd2 regulates plant innate immunity through multiple interconnected mechanisms, requiring sophisticated experimental approaches to fully characterize:

Methodological approach: Compare wild-type and Fd2-KO plants challenged with virulent pathogens, avirulent pathogens, and purified PAMPs using bacterial growth assays, disease rating scales, and qRT-PCR of defense marker genes.

Hormone Signaling Integration:
Upon Pst DC3000 infection, Fd2-KO mutants accumulate increased levels of jasmonic acid while showing compromised salicylic acid-related immune responses . This suggests Fd2 helps modulate the balance between these antagonistic defense hormone pathways.

Methodological approach: Quantify hormone levels using LC-MS/MS in wild-type versus Fd2-KO plants at different timepoints after infection. Combine with transcript analysis of JA- and SA-responsive marker genes and genetic crosses with hormone signaling mutants.

ROS Signaling:
Fd2-KO mutants show defects in reactive oxygen species (ROS) accumulation during PTI responses , implicating Fd2 in early immune signaling events.

Methodological approach: Measure ROS production using luminol-based assays or specific fluorescent probes in leaf discs treated with PAMPs. Compare subcellular ROS distribution using confocal microscopy with appropriate ROS-sensitive fluorescent indicators.

Protein Interactions:
Fd2 physically interacts with FIBRILLIN4 (FIB4), a harpin-binding protein localized in chloroplasts , suggesting formation of functional complexes involved in immune regulation.

Methodological approach: Perform co-immunoprecipitation with Anti-Fd2 antibodies followed by Western blot detection of FIB4. Confirm interactions using bimolecular fluorescence complementation or proximity ligation assays in planta. Identify additional interacting partners using immunoprecipitation coupled with mass spectrometry.

Unique Localization:
Unlike its interacting partner FIB4, Fd2 localizes to stromules extending from chloroplasts , potentially facilitating signal transmission between cellular compartments during immune responses.

Methodological approach: Use immunofluorescence with Anti-Fd2 antibodies combined with chloroplast markers to visualize localization. Track stromule dynamics and Fd2 localization during pathogen infection using time-lapse imaging in transgenic lines expressing fluorescently-tagged Fd2.

These methodological approaches, often used in combination, have revealed Fd2's complex role at the intersection of photosynthetic electron transport and immune signaling networks.

What techniques can be used to study FD2's protein-protein interactions, and how can antibodies facilitate these analyses?

Understanding Fd2's protein-protein interactions is crucial for deciphering its role in plant immunity and photosynthesis. Several methodological approaches utilizing Fd2 antibodies can effectively characterize these interactions:

Co-immunoprecipitation (Co-IP):

• Methodology: Solubilize plant tissue in extraction buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0) , then immunoprecipitate using Anti-Fd2 antibodies coupled to protein A/G beads. Analyze precipitated complexes by Western blotting with antibodies against suspected interacting partners or by mass spectrometry for unbiased discovery.

• Application: This approach has successfully identified the interaction between Fd2 and FIBRILLIN4 (FIB4) and can be used to discover novel Fd2-interacting proteins.

• Validation: Include negative controls such as pre-immune serum or IgG from non-immunized animals, and positive controls with known interactors.

Proximity Ligation Assay (PLA):

• Methodology: Fix plant tissue sections, incubate with Anti-Fd2 antibodies alongside antibodies against potential interacting partners, followed by species-specific PLA probes, ligation, and rolling circle amplification.

• Application: Visualize Fd2 interactions with spatial resolution in situ, confirming interactions occur in their native cellular context.

• Advantage: Can detect low-abundance interactions and provides subcellular localization information.

Bimolecular Fluorescence Complementation (BiFC) with Antibody Validation:

• Methodology: Express Fd2 and candidate interactors fused to complementary fragments of a fluorescent protein. Confirm interactions detected by BiFC using co-immunoprecipitation with Anti-Fd2 antibodies.

• Application: Visualize interactions in living cells while providing information about subcellular localization.

• Validation: Quantify signal intensity and compare with appropriate negative controls (non-interacting proteins).

Antibody-based Pull-down of Recombinant Proteins:

• Methodology: Express recombinant Fd2 and potential interacting partners in vitro, perform pull-down assays with Anti-Fd2 antibodies, and analyze binding through Western blotting.

• Application: Test direct physical interactions and map interaction domains by using truncated protein variants.

• Controls: Include competitive binding assays with excess untagged protein to confirm specificity.

Subcellular Co-localization Studies:

• Methodology: Perform double immunofluorescence labeling with Anti-Fd2 antibodies and antibodies against potential interacting partners.

• Application: Determine whether proteins share the same subcellular location, such as Fd2's unique localization to stromules extending from chloroplasts .

• Enhancement: Combine with super-resolution microscopy techniques for higher precision spatial information.

A comprehensive approach combining these techniques can provide robust evidence for Fd2's interaction network, offering insights into its functional roles in both photosynthesis and immune regulation.

How does FD2 expression and localization change during pathogen infection, and what methodological approaches can track these changes?

Tracking changes in Fd2 expression and localization during pathogen infection requires integrating multiple experimental approaches, with antibodies serving as critical tools for detection and analysis:

Expression Analysis:
Research has established that Fd2 expression is suppressed upon infection with Pseudomonas syringae pv. tomato (Pst) DC3000 , suggesting pathogen-mediated manipulation of this chloroplastic protein.

Quantitative methods:

• Western blot time course: Use Anti-Fd2 antibodies (1:1000-1:5000 dilution) to quantify Fd2 protein levels at different timepoints after pathogen inoculation.

• RT-qPCR: Complement protein analysis with transcript measurements to determine if suppression occurs at transcriptional or post-transcriptional levels.

• In situ protein analysis: Immunohistochemistry with Anti-Fd2 antibodies to visualize tissue-specific expression changes during infection progression.

Localization Dynamics:
Fd2 uniquely localizes to both chloroplasts and stromules (stroma-filled tubular extensions) , with potential redistribution during immune responses.

Visualization techniques:

• Confocal immunofluorescence: Fixed tissue sections labeled with Anti-Fd2 antibodies and fluorescent secondary antibodies, combined with chloroplast markers to track changes in distribution patterns.

• Subcellular fractionation: Separate chloroplasts, stromules, and other cellular compartments via differential centrifugation, followed by Western blot analysis with Anti-Fd2 antibodies to quantify redistribution.

• Live-cell imaging: Transgenic plants expressing fluorescently-tagged Fd2, validated by immunofluorescence with Anti-Fd2 antibodies, to monitor real-time changes during infection.

Stromule dynamics analysis:
Since Fd2 localizes to stromules unlike its interacting partner FIB4 , tracking stromule formation and Fd2 distribution within these structures during infection provides important insights.

Methodological approach:

• Dual-channel confocal microscopy using Anti-Fd2 antibodies and stromule markers

• Quantification of stromule number, length, and Fd2 content before and after pathogen challenge

• Three-dimensional reconstruction to visualize spatial relationships between chloroplasts, stromules, and pathogen structures

Experimental design considerations:

• Include appropriate controls (mock-inoculated plants, non-specific antibody controls)

• Compare virulent and avirulent pathogen strains to distinguish PTI and ETI responses

• Use time-course experiments starting from early infection (minutes) to later stages (days)

• Compare localization patterns in wild-type versus defense signaling mutants to establish pathway dependencies

These methodological approaches collectively provide a comprehensive view of how pathogens impact both the abundance and subcellular distribution of Fd2, offering insights into chloroplast-mediated immune responses.

What are the recommended protocols for detecting post-translational modifications of FD2 using antibodies?

While the search results don't directly address detection of post-translational modifications (PTMs) of Fd2, researchers can implement several antibody-based methodological approaches to investigate potential PTMs:

Two-dimensional gel electrophoresis with immunoblotting:

• Separate plant protein extracts by isoelectric focusing in the first dimension

• Perform SDS-PAGE in the second dimension

• Transfer to membrane and probe with Anti-Fd2 antibodies (1:1000-1:5000 dilution)

• Identify potential PTM-modified Fd2 by shifts in isoelectric point or apparent molecular weight

• Compare patterns before and after treatment with specific modification-removing enzymes (phosphatases, deglycosylases, etc.)

Immunoprecipitation coupled with mass spectrometry:

• Extract plant proteins under non-denaturing conditions using buffer containing 150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl (pH 8.0)

• Immunoprecipitate Fd2 using Anti-Fd2 antibodies

• Process immunoprecipitated material for LC-MS/MS analysis

• Analyze MS/MS spectra for signature mass shifts indicating specific PTMs

• Confirm findings using targeted MS approaches such as selected reaction monitoring

Modification-specific antibodies in combination with Anti-Fd2:

• Immunoprecipitate Fd2 using Anti-Fd2 antibodies

• Probe western blots of immunoprecipitates with antibodies specific for common PTMs:

  • Anti-phosphoserine/threonine/tyrosine antibodies

  • Anti-ubiquitin antibodies

  • Anti-SUMO antibodies

  • Anti-acetyllysine antibodies

• Confirm positive signals with appropriate controls (phosphatase treatment, etc.)

Phos-tag™ SDS-PAGE for phosphorylation analysis:

• Prepare plant extracts with phosphatase inhibitors

• Run parallel samples on standard SDS-PAGE and Phos-tag™-containing gels

• Transfer and immunoblot with Anti-Fd2 antibodies (1:1000-1:5000)

• Identify phosphorylated forms by mobility shifts specific to Phos-tag™ gels

• Confirm by treating duplicate samples with phosphatase before electrophoresis

Native gel electrophoresis for redox state analysis:

• Extract proteins under non-reducing conditions

• Perform native PAGE with and without prior treatment with reducing agents

• Transfer and immunoblot with Anti-Fd2 antibodies

• Identify redox-dependent mobility shifts indicating oxidation states of Fd2

Experimental considerations:

• Always include appropriate controls (enzyme treatments, mutants in PTM machinery)

• Consider stress conditions that might induce PTMs (pathogen infection, oxidative stress)

• Compare wild-type plants with Fd2-KO mutants to confirm band specificity

• For pathogen-responsive studies, analyze samples at multiple timepoints after infection

These methodological approaches provide a systematic framework for identifying and characterizing potential PTMs of Fd2, which may play important roles in regulating its function in photosynthesis and immune responses.

How can FD2 antibodies be incorporated into multi-omics approaches for comprehensive plant immunity studies?

Integrating Fd2 antibodies into multi-omics approaches creates powerful experimental frameworks for comprehensive understanding of plant immunity. Here are methodological strategies combining antibody-based techniques with other omics platforms:

Proteomics integration:

• Immunoprecipitation-Mass Spectrometry (IP-MS)

  • Methodology: Immunoprecipitate Fd2 protein complexes using Anti-Fd2 antibodies from plants at different stages of infection

  • Analyze by tandem mass spectrometry to identify infection-specific interacting partners

  • Validate key interactions using reciprocal co-immunoprecipitation and Western blotting

  • Data analysis: Compare protein interaction networks between healthy and infected plants

• Spatial proteomics with antibody validation

  • Methodology: Fractionate plant cells into subcellular compartments

  • Perform proteomics analysis on fractions and validate Fd2 distribution using immunoblotting

  • Track changes in Fd2 compartmentalization during infection

  • Data integration: Correlate with changes in other chloroplast-localized immune components

Transcriptomics correlation:

• ChIP-seq adjacent methodology

  • Methodology: Perform RNA-seq on wild-type and Fd2-KO plants before and after pathogen challenge

  • Compare with protein levels detected by Anti-Fd2 immunoblotting

  • Identify genes differentially expressed in Fd2-dependent manner

  • Data validation: Confirm selected transcriptional changes using RT-qPCR and correlate with Fd2 protein levels

Metabolomics linkage:

• Antibody-based enzyme activity assays

  • Methodology: Immunoprecipitate Fd2 to measure associated enzyme activities

  • Correlate with metabolomic profiles of wild-type versus Fd2-KO plants

  • Focus on metabolites in redox-sensitive pathways potentially affected by altered electron transfer

  • Data interpretation: Establish connections between Fd2 activity and metabolic signatures of immunity

Phenomics correlation:

• High-throughput phenotyping with biochemical validation

  • Methodology: Perform automated phenotyping of plant responses to pathogens

  • Correlate visible phenotypes with Fd2 protein levels determined by quantitative immunoblotting

  • Generate response curves relating Fd2 abundance to disease resistance phenotypes

  • Data modeling: Develop predictive models of how Fd2 levels influence disease outcomes

Multi-scale imaging approaches:

• Correlative microscopy with immunolabeling

  • Methodology: Combine immunofluorescence using Anti-Fd2 antibodies with electron microscopy

  • Visualize Fd2 distribution at both tissue and ultrastructural levels during infection

  • Integrate with live-cell imaging of defense responses

  • Image analysis: Quantify spatial relationships between Fd2, chloroplasts, and pathogen structures

Computational integration:

• Network biology with experimental validation

  • Methodology: Build protein interaction networks based on immunoprecipitation data

  • Integrate with transcriptomic and metabolomic datasets

  • Identify network modules regulated by Fd2

  • Experimental validation: Test predictions using targeted Fd2 antibody-based assays

These integrated approaches provide a systems-level understanding of how Fd2 functions within the complex web of plant immune responses, connecting molecular mechanisms to cellular processes and ultimately to whole-plant phenotypes.

What are the potential limitations and technical challenges when using FD2 antibodies, and how can researchers address them?

When using Fd2 antibodies in research, several technical challenges and limitations may arise. Here are methodological approaches to identify and address these issues:

Cross-reactivity with related ferredoxin isoforms:

Ferredoxin proteins share significant sequence homology, potentially leading to antibody cross-reactivity. The polyclonal Anti-Fd2 antibody described in the search results was raised against full-length maize Fd2 , which may recognize conserved epitopes present in multiple ferredoxin isoforms.

Methodological solutions:

• Validation using knockout controls: Always include Fd2-knockout plant samples as negative controls in immunoblotting experiments

• Pre-absorption strategy: Incubate antibodies with recombinant proteins of potential cross-reacting ferredoxin isoforms before use in experiments

• Epitope mapping: Determine which regions of Fd2 are recognized by the antibody to predict potential cross-reactivity

• Isoform-specific peptide competition: Use synthetic peptides unique to Fd2 to confirm signal specificity

Detection sensitivity limitations:

Fd2 may be expressed at varying levels depending on tissue type, developmental stage, or stress conditions, potentially falling below detection thresholds in certain samples.

Methodological solutions:

• Signal amplification techniques: Employ tyramide signal amplification for immunofluorescence or enhanced chemiluminescence for Western blots

• Sample enrichment: Perform chloroplast isolation before immunoblotting to concentrate Fd2

• Optimized extraction buffers: Develop buffers specifically designed for efficient Fd2 extraction (consider the buffer containing 150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0 mentioned in the search results)

• Loading control optimization: Use chloroplast-specific loading controls rather than whole-cell controls when analyzing Fd2 levels

Fixation-sensitive epitopes:

Fd2's chloroplastic and stromule localization may require specific fixation protocols that preserve epitope recognition while maintaining structural integrity.

Methodological solutions:

• Fixation optimization: Test multiple fixation protocols (paraformaldehyde, glutaraldehyde, methanol) at various concentrations and durations

• Antigen retrieval: Develop specific antigen retrieval methods for Fd2 in fixed tissues

• Live-cell antibody approaches: Consider membrane-permeable antibody fragments for live-cell imaging

• Correlative validation: Confirm immunolabeling patterns with fluorescently-tagged Fd2 expression

Batch-to-batch variability:

The polyclonal nature of the Anti-Fd2 antibody may lead to variation between production batches.

Methodological solutions:

• Antibody validation protocols: Establish standardized testing protocols for each new antibody batch

• Reference sample banking: Maintain a set of reference samples with known Fd2 levels for calibration

• Monoclonal alternative investigation: Consider developing monoclonal antibodies for applications requiring higher consistency

• Antibody pooling strategy: Pool multiple production batches to minimize variation in long-term studies

Pathogen-induced epitope masking:

Since Fd2 expression is suppressed during pathogen infection , pathogen-induced modifications or interactions might mask epitopes recognized by the antibody.

Methodological solutions:

• Multiple antibody approach: Use antibodies recognizing different Fd2 epitopes

• Denaturing conditions optimization: Test various sample preparation conditions to expose potentially masked epitopes

• Native vs. denatured comparison: Compare results from native PAGE and denaturing SDS-PAGE immunoblots

• Time-course analysis: Track early infection events before significant protein level changes occur

By implementing these methodological solutions, researchers can overcome technical challenges and generate more reliable and reproducible data when using Fd2 antibodies in their experimental systems.