At5g45490 Antibody

What is AT5G45490 and why is it significant in plant research?

AT5G45490 is a gene in Arabidopsis thaliana that belongs to Group C of the coiled-coil nucleotide-binding leucine-rich repeat (CC-NLR) family of plant immune receptors. This gene encodes a protein involved in plant immunity and stress responses.
The significance of AT5G45490 stems from its role in plant defense mechanisms. As part of the CC-NLR family, it contributes to the plant's ability to recognize pathogen effectors and trigger immune responses. Research on AT5G45490 provides insights into how plants detect and respond to pathogen attacks, which is crucial for developing disease-resistant crops .

How are antibodies against AT5G45490 typically generated?

Antibodies against AT5G45490 are typically generated through one of several approaches:

• Peptide-based immunization: Synthetic peptides corresponding to unique regions of the AT5G45490 protein are conjugated to carrier proteins (like KLH) and used to immunize rabbits or other animals. This approach is commonly used when targeting specific epitopes .

• Recombinant protein immunization: The AT5G45490 protein or specific domains are expressed in bacterial systems (typically E. coli), purified, and used as immunogens. This method is advantageous when conformational epitopes are important .

• Genetic immunization: DNA encoding AT5G45490 is introduced into an animal, resulting in in vivo expression and immune response against the native conformation of the protein.
  For monoclonal antibodies, additional steps include hybridoma generation or display technologies (phage, yeast, or mammalian display) to select high-affinity binders .

What are the essential validation steps for an AT5G45490 antibody?

A robust validation strategy for AT5G45490 antibodies should include:

How can I differentiate between AT5G45490 and other closely related NLR proteins?

Distinguishing AT5G45490 from closely related NLR proteins requires careful consideration of:

• Epitope selection: Choose unique regions that differ from homologous proteins. Typically, the N-terminal region or variable loops in the LRR domain show greater sequence divergence .

• Cross-reactivity testing: Test the antibody against recombinant proteins of close homologs, particularly AT5G45440 and AT4G19060, which are placed in the same phylogenetic group .

• Immunoblotting patterns: AT5G45490 will produce a distinct band pattern that can be compared with predicted molecular weights of related proteins. Running parallel samples from plants expressing tagged versions of different NLRs can help identify specific bands .

• Immunoprecipitation followed by mass spectrometry: This can definitively identify which protein(s) the antibody is recognizing .

• Epitope mapping: Determine the exact binding site of the antibody to confirm specificity to AT5G45490-unique regions .

What are the optimal conditions for using AT5G45490 antibodies in Western blotting?

Optimal Western blotting conditions for AT5G45490 antibodies typically include:

• Sample preparation:

  • Extract proteins using a TCA-acetone method for higher protein yield from plant tissues

  • Add protease inhibitors to prevent degradation

  • Include phosphatase inhibitors if studying phosphorylation states

• Gel conditions:

  • Use 10-12% SDS-PAGE for optimal resolution of AT5G45490 (expected MW: ~100-120 kDa)

  • Consider gradient gels (4-20%) if analyzing protein complexes

• Transfer parameters:

  • Semi-dry transfer at 7 minutes for PVDF membranes (0.2 μm pore size)

  • Alternatively, wet transfer at 30V overnight for larger proteins

• Blocking and antibody incubation:

  • Block with 5% milk in TBS-T for 4°C overnight

  • Primary antibody dilution: 1:500 to 1:1000 in TBS-T

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

  • Three 15-minute washes in TBS-T

• Detection:

  • HRP-conjugated secondary antibodies at 1:10,000 dilution

  • ECL detection system with exposure times optimized for signal intensity

How can AT5G45490 antibodies be used for chromatin immunoprecipitation (ChIP) studies?

For ChIP applications with AT5G45490 antibodies:

• Cross-linking protocol:

  • Two-week-old Arabidopsis seedlings in liquid MS medium

  • Treat with 1% formaldehyde for 10 minutes under vacuum

  • Quench with 0.125 M glycine for 5 minutes

• Chromatin preparation:

  • Isolate nuclei using sucrose gradient centrifugation

  • Sonicate to achieve fragments of 200-500 bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A beads

  • Incubate with AT5G45490 antibody (5-10 μg) overnight at 4°C

  • Include appropriate controls: IgG negative control and a positive control antibody (e.g., anti-histone H3)

• Washing and elution:

  • Wash with increasing stringency buffers (low salt, high salt, LiCl, TE)

  • Elute with SDS buffer at 65°C

  • Reverse crosslinks and purify DNA

• Analysis:

  • Perform qPCR targeting potential binding regions

  • Use primers designed to amplify 80-150 bp regions
    Note that this application would be relevant if AT5G45490 is involved in DNA binding or chromatin association, which would need to be established first through localization studies.

What strategies can overcome weak or non-specific signals when using AT5G45490 antibodies?

When encountering weak or non-specific signals:

• For weak signals:

  • Increase antibody concentration (up to 1:200 for immunofluorescence)

  • Optimize antigen retrieval (for fixed tissues): try heat-induced epitope retrieval with citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems (TSA/CARD)

  • Optimize extraction buffers to ensure solubilization of membrane-bound proteins

• For non-specific signals:

  • Increase blocking stringency (5% BSA or 10% normal serum)

  • Add detergents (0.1-0.3% Triton X-100) to reduce hydrophobic interactions

  • Perform affinity purification of the antibody against the immunizing peptide

  • Pre-adsorb antibody with plant extracts from AT5G45490 knockout lines

  • Reduce secondary antibody concentration

• For high background:

  • Include additional washing steps with higher salt concentration (up to 500 mM NaCl)

  • Add 0.1% SDS or 0.5% deoxycholate to wash buffers for more stringent conditions

  • Use specialized blocking reagents (commercial blockers with irrelevant proteins)

How can AT5G45490 antibodies be used to study protein-protein interactions in immune signaling pathways?

For studying protein-protein interactions involving AT5G45490:

• Co-immunoprecipitation (Co-IP):

  • Extract proteins under native conditions (avoid harsh detergents)

  • Use 100-300 mM NaCl to maintain interactions while reducing non-specific binding

  • Cross-link weak or transient interactions using DSP or formaldehyde

  • Analyze precipitated complexes by immunoblotting or mass spectrometry

  • Compare samples with and without immune stimulation to identify dynamic interactions

• Proximity ligation assay (PLA):

  • Use AT5G45490 antibody with antibodies against potential interacting partners

  • Perform in fixed Arabidopsis tissues or protoplasts

  • Analyze discrete fluorescent spots indicating proximity (<40 nm)

  • Quantify interaction frequencies under different conditions

• Bimolecular fluorescence complementation (BiFC) validation:

  • Clone AT5G45490 and potential interactors identified by Co-IP into BiFC vectors

  • Express in Arabidopsis protoplasts or N. benthamiana leaves

  • Confirm interactions observed with antibody-based methods

• Immunogold electron microscopy:

  • Precisely localize AT5G45490 at the ultrastructural level

  • Perform double-labeling with different sized gold particles to visualize co-localization

  • Analyze spatial relationships of immune complexes during different stages of pathogen response

What considerations are important when using AT5G45490 antibodies for developmental studies across different plant tissues?

For developmental studies using AT5G45490 antibodies:

• Tissue-specific optimization:

  • Different fixation protocols for various tissues (roots require shorter fixation than leaves)

  • Tissue-specific permeabilization: 0.1% Triton X-100 for leaves vs. 0.5% for roots

  • Antigen retrieval may be necessary for highly lignified tissues

• Developmental stage considerations:

  • Protein expression levels may vary dramatically across developmental stages

  • Adjust antibody concentrations accordingly (higher for tissues with lower expression)

  • Include stage-specific positive controls known to express AT5G45490

• Comparative analysis across tissues:

  • Use internal loading controls appropriate for each tissue type

  • Normalize signals to account for tissue-specific extraction efficiencies

  • Consider tissue-specific post-translational modifications affecting epitope recognition

• Technical accommodations:

  • For thick tissues: increase incubation times and use vacuum infiltration

  • For autofluorescent tissues: use appropriate emission filters or spectral unmixing

  • For tissues with high phenolic content: add PVP or PVPP to extraction/incubation buffers

• Validation approaches:

  • Generate transgenic lines expressing fluorescently-tagged AT5G45490 under native promoter

  • Compare antibody staining patterns with reporter gene expression

  • Perform RNA in situ hybridization as complementary localization method

How can phospho-specific antibodies against AT5G45490 be developed to study its activation state?

Developing phospho-specific antibodies for AT5G45490 involves:

• Identification of phosphorylation sites:

  • Perform mass spectrometry analysis of immunoprecipitated AT5G45490 from plants under normal and pathogen-challenged conditions

  • Use phospho-proteomics databases and prediction algorithms to identify potential regulatory phosphorylation sites

  • Focus on conserved motifs in NLR proteins known to regulate activity (e.g., P-loop region)

• Peptide design for immunization:

  • Generate synthetic phosphopeptides (10-15 amino acids) containing the phosphorylated residue in the center

  • Include a C-terminal cysteine for conjugation if not naturally present

  • Synthesize both phosphorylated and non-phosphorylated versions of the same peptide

• Antibody production strategy:

  • Immunize animals with the phosphopeptide conjugated to KLH or another carrier

  • Perform dual-affinity purification:
    a. Positive selection on phosphopeptide column
    b. Negative selection on non-phosphopeptide column to remove antibodies recognizing the non-phosphorylated form

• Validation of phospho-specificity:

  • Western blot comparing samples treated with and without phosphatase

  • Peptide competition assays with phospho and non-phospho peptides

  • Immunoprecipitation followed by MS to confirm phosphorylation status of bound proteins

• Application in signaling studies:

  • Monitor phosphorylation kinetics during immune activation

  • Identify stimuli that trigger AT5G45490 phosphorylation

  • Map signaling pathways using specific kinase inhibitors

What are the considerations for developing conformational antibodies that specifically recognize activated AT5G45490?

Developing conformation-specific antibodies requires:

• Understanding structural changes:

  • NLR proteins undergo substantial conformational changes upon activation

  • Focus on regions exposed only in the active state (e.g., nucleotide-binding domain after ADP-ATP exchange)

  • Consider epitopes at domain interfaces that become accessible upon oligomerization

• Immunization strategies:

  • Use full-length protein locked in active conformation (e.g., ATP-γ-S bound)

  • Alternatively, use domain fragments that mimic the active conformation

  • Consider nanobody development, as single-domain antibodies often recognize conformational epitopes

• Screening methodology:

  • Develop assays that can distinguish active vs. inactive states

  • Use differential ELISA with protein prepared under activating vs. non-activating conditions

  • Employ microscale thermophoresis to measure binding to different conformational states

• Validation approaches:

  • Immunoprecipitation under native conditions with and without activating ligands

  • Structural studies (cryo-EM, X-ray) of antibody-antigen complexes

  • Functional assays to confirm antibody binding correlates with activity

• Applications in research:

  • Real-time monitoring of AT5G45490 activation in live cells

  • Stabilization of active conformation for structural studies

  • Selective inhibition of active vs. inactive protein pools
    This conformational antibody would be invaluable for studying the activation mechanisms of plant NLRs and could serve as a tool for screening compounds that modulate immune responses.

How do recombinant expression systems compare for producing AT5G45490 antigens for antibody development?

Comparison of expression systems for AT5G45490 antigens:

What methods can detect rare conformational states of AT5G45490 during immune activation?

Advanced methods for detecting rare conformational states:

• Single-molecule FRET combined with antibody labeling:

  • Label different domains with donor/acceptor fluorophores

  • Use conformation-specific antibodies to stabilize rare states

  • Monitor conformational dynamics in real-time

  • Quantify the population of different conformational states

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with antibody capture:

  • Use antibodies to capture specific conformational states

  • Perform HDX-MS to map solvent accessibility changes

  • Identify regions involved in conformational transitions

  • Protocol considerations:

    • Short labeling times (10s-1min) to capture transient states

    • Rapid quenching to prevent back-exchange

    • Online pepsin digestion for spatial resolution

• Cryo-electron microscopy with antibody fragments:

  • Use Fab fragments to stabilize specific conformations

  • Apply single-particle cryo-EM analysis to resolve 3D structures

  • Identify conformational epitopes and structural transitions

  • Compare structures from plants at different activation states

• Native mass spectrometry:

  • Preserve non-covalent interactions during ionization

  • Distinguish between monomeric and oligomeric states

  • Detect nucleotide binding and exchange

  • Monitor complex formation with signaling partners

• Cross-linking mass spectrometry with activation-specific antibodies:

  • Cross-link proteins in different activation states

  • Immunoprecipitate specific conformers with antibodies

  • Identify distance constraints through MS analysis

  • Map structural changes during activation These methods would provide unprecedented insights into the conformational dynamics of AT5G45490 during immune signaling, potentially revealing novel therapeutic targets for plant disease resistance.