At1g68907 Antibody

What is the At1g68907 gene and why are antibodies against its protein product important in plant defense research?

At1g68907 encodes a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor involved in defense response pathways similar to the well-characterized SlNAC1 in tomato. Antibodies against this protein are critical for investigating plant immune responses, particularly those involving transcriptional reprogramming during pathogen detection. The protein functions in a regulatory network that balances defense activation with preventing costly autoimmunity in the absence of pathogens . Methodologically, these antibodies enable protein localization studies, protein-protein interaction analyses, and investigation of post-translational modifications that regulate defense-related transcription factor activity.

What are the recommended immunoprecipitation protocols when working with At1g68907 antibody?

For optimal immunoprecipitation of At1g68907 protein and its interacting partners, researchers should:

• Harvest and flash-freeze 2-3g of plant tissue in liquid nitrogen

• Grind tissue with mortar and pestle while maintaining freezing conditions

• Extract proteins in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 0.5% Triton X-100, 1mM EDTA, 1mM DTT, and protease inhibitor cocktail

• Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Incubate cleared lysate with At1g68907 antibody (typically 2-5μg) overnight at 4°C

• Add protein A/G matrix and incubate for 2-3 hours at 4°C

• Wash beads 4-5 times with extraction buffer containing reduced detergent (0.1%)

• Elute proteins with SDS sample buffer or low pH buffer

For studying protein-protein interactions, a two-step immunoprecipitation similar to the MBP-SlNAC1-HA purification protocol can be adapted, where the protein complex is initially immunoprecipitated with anti-HA antibody matrix followed by Western blotting with anti-FLAG antibody to detect interacting proteins .

How can I validate the specificity of an At1g68907 antibody for immunoblotting applications?

Validating antibody specificity requires multiple complementary approaches:

• Genetic Controls: Compare wild-type plants with At1g68907 knockout/knockdown lines to confirm the absence of signal in mutant lines

• Overexpression Controls: Test the antibody against samples from plants overexpressing At1g68907 with epitope tags

• Peptide Competition Assay: Pre-incubate the antibody with excess synthetic peptide corresponding to the epitope to block specific binding

• Cross-reactivity Assessment: Test antibody against recombinant At1g68907 protein alongside related NAC transcription factors to ensure specificity

When performing immunoblotting, use 10-12% SDS-PAGE gels for optimal resolution, transfer to PVDF membranes at 100V for 60-90 minutes, and block with 5% non-fat milk or BSA for at least 1 hour before antibody incubation. The most effective antibody dilutions typically range from 1:1000 to 1:5000 depending on the specific antibody preparation .

How can I optimize chromatin immunoprecipitation (ChIP) protocols using At1g68907 antibody to identify direct transcriptional targets?

ChIP optimization for At1g68907 requires careful consideration of multiple technical parameters:

• Crosslinking Optimization: Test both 1% formaldehyde for 10 minutes and dual crosslinking with 1.5mM EGS followed by 1% formaldehyde for improved protein-DNA fixation

• Sonication Parameters: Optimize sonication to yield DNA fragments of 200-500bp (typically 12-15 cycles of 30sec on/30sec off at 40% amplitude)

• Antibody Specificity: Validate ChIP-grade quality of the At1g68907 antibody using known targets or tagged protein controls

• Negative Controls: Include no-antibody controls and IgG controls alongside At1g68907 antibody samples

• Positive Controls: Include antibodies against general transcription factors or histone modifications as technical success indicators

For identifying direct targets, interrogate precipitated DNA using qPCR for predicted NAC recognition sequences (NACRS) in promoters of defense-related genes. NAC transcription factors typically recognize the core motif CAT(G/A)T(G/A) in the promoters of target genes, similar to binding sites identified for SlNAC1 in defense response genes .

What approaches can resolve contradictory data regarding At1g68907 protein localization or interaction partners?

When faced with contradictory data regarding At1g68907 localization or interactions, implement the following methodological strategies:

• Multiple Localization Techniques: Compare results from:

  • Antibody-based immunofluorescence

  • Fluorescent protein fusions (both N- and C-terminal)

  • Cell fractionation followed by immunoblotting

  • Proximity ligation assays

• Protein Interaction Verification Matrix:

• Investigate Post-translational Modifications: PTMs like phosphorylation, ubiquitination or SUMOylation can dramatically affect localization and interaction patterns. Analyze the protein under different stress conditions that might trigger defense responses, similar to how SlNAC1 regulation changes during pathogen recognition .

How can antibody affinity extraction (AAE) be applied to improve At1g68907 antibody coverage evaluation?

Antibody affinity extraction provides superior coverage evaluation compared to traditional methods like 2D Western blotting. For At1g68907 antibody assessment:

• Column Preparation:

  • Immobilize purified At1g68907 antibody covalently onto chromatography support

  • Condition the column to prevent antibody leaching and minimize nonspecific binding

  • Validate column efficiency with known quantities of recombinant At1g68907

• Sample Processing:

  • Pass native, undenatured plant extract containing At1g68907 over the column

  • Elute bound proteins with acid buffer (typically glycine-HCl, pH 2.5)

  • Cycle the sample repeatedly over the column until no additional protein is bound

  • Pool elution fractions, neutralize, and concentrate to original sample volume

• Analysis Options:

  • Subject the pre-AAE and post-AAE samples to 2D-PAGE or mass spectrometric analysis

  • Quantify depletion of At1g68907 and its interacting proteins

  • Identify specifically-bound versus non-specifically-bound proteins

This approach allows for quantitative assessment of antibody reactivity to the most important proteins, particularly those that copurify with At1g68907 under different experimental conditions .

How should I address epitope masking issues when At1g68907 forms complexes with other defense-related proteins?

Epitope masking occurs frequently with transcription factors like At1g68907 that participate in multiple protein complexes. To overcome this challenge:

• Epitope Mapping: Determine which regions of At1g68907 are recognized by the antibody through peptide arrays or deletion constructs

• Mild Denaturation: Test varying concentrations of SDS (0.1-1%) or urea (1-4M) in extraction buffers to partially denature complexes without destroying the epitope

• Multiple Antibody Approach: Develop antibodies against different regions of At1g68907 to ensure detection regardless of complex formation

• Complex Dissociation Protocols:

  • Heat samples to 65°C (not 95°C) in the presence of 1% SDS

  • Include 10mM DTT to reduce disulfide bonds

  • Sonicate briefly to disrupt protein-protein interactions

For plant defense-related transcription factors like At1g68907, it's important to consider that protein interactions may change dramatically upon pathogen challenge, similar to how SlNAC1 interactions are regulated by the Prf protein to prevent its degradation by SlSINA3 during immune responses .

What strategies can optimize detection of post-translational modifications of At1g68907 during defense responses?

Post-translational modifications critically regulate NAC transcription factors like At1g68907 during defense responses. To effectively study these modifications:

• Phosphorylation Analysis:

  • Treat samples with phosphatase inhibitors (50mM NaF, 10mM Na₃VO₄) during extraction

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Employ phospho-specific antibodies if phosphorylation sites are known

  • Consider combining with mass spectrometry for site identification

• Ubiquitination Detection:

  • Include deubiquitinase inhibitors (5mM N-ethylmaleimide) in extraction buffers

  • Perform immunoprecipitation under denaturing conditions (1% SDS, 95°C)

  • Probe with anti-ubiquitin antibodies after At1g68907 immunoprecipitation

  • Compare ubiquitination patterns before and after pathogen challenge

• SUMOylation Assessment:

  • Extract proteins in the presence of SUMO protease inhibitors (20mM N-ethylmaleimide)

  • Detect with anti-SUMO antibodies after At1g68907 immunoprecipitation

  • Mutate predicted SUMOylation sites to confirm specificity

The dynamic interplay between these modifications likely regulates At1g68907 stability and function, similar to how SlNAC1 stability is regulated through the interaction between SlSINA3 (an E3 ubiquitin ligase) and activated Prf to balance defense activation with preventing autoimmunity .

How can I study the dynamics of At1g68907 protein-protein interactions during different stages of the plant immune response?

Studying the temporal dynamics of At1g68907 interactions during immune responses requires synchronized experimental systems and time-resolved analyses:

• Synchronized Immune Stimulation:

  • Use PAMPs like flg22 or effector proteins for precise timing of immune activation

  • Establish time-course sampling at key stages (0, 15, 30, 60, 180 minutes, 24 hours)

  • Include controls for SA and JA pathway activation status at each timepoint

• Sequential Co-immunoprecipitation:

  • Perform At1g68907 immunoprecipitation at each time point

  • Analyze interaction partners by mass spectrometry or targeted immunoblotting

  • Use label-free quantification or SILAC to measure changes in interaction strength

• Live-Cell Imaging:

  • Generate split fluorescent protein fusions to visualize interactions in real-time

  • Employ photoactivatable or photoconvertible tags to track newly synthesized versus existing proteins

  • Correlate protein interaction dynamics with transcriptional outputs

• Data Analysis Framework:

This approach allows for mapping the complete interactome dynamics of At1g68907 during defense responses, similar to studies on SlNAC1 that revealed its sequestration away from the E3 ubiquitin ligase SlSINA3 by activated Prf during immune activation .

How might monoclonal antibodies against At1g68907 improve detection specificity compared to polyclonal antibodies?

Monoclonal antibodies offer several methodological advantages for At1g68907 research:

• Epitope-Specific Recognition: Unlike polyclonal antibodies that recognize multiple epitopes, monoclonal antibodies bind to a single epitope, allowing for consistent specificity across experiments and batches

• Reduced Background: Monoclonal antibodies typically produce cleaner Western blots and immunofluorescence images by eliminating cross-reactivity with related NAC family members

• Improved Reproducibility: The homogeneous nature of monoclonal antibodies ensures consistent performance across different research groups and over time

Development of monoclonal antibodies against At1g68907 would follow similar approaches to those used for important viral proteins, where researchers identified specific target regions for antibody development . The process involves:

• Immunizing mice with recombinant At1g68907 protein or synthetic peptides

• Screening hybridoma clones for specific binding to At1g68907

• Selecting clones that recognize functionally important domains (DNA-binding, protein interaction, or regulatory regions)

• Validating specificity through knockout controls and cross-reactivity testing

Each monoclonal antibody would be characterized for its specific epitope and optimal application conditions, similar to how researchers developed mAbs targeting specific proteins on Epstein-Barr virus .

What are the best approaches for developing At1g68907 antibodies that can distinguish between different post-translationally modified forms?

Developing modification-specific antibodies requires strategic planning:

• Modification Site Identification:

  • Perform mass spectrometry analysis of At1g68907 under different conditions

  • Map modification sites to functional domains

  • Prioritize conserved modifications found in related NAC proteins

• Modified Peptide Design:

  • Synthesize peptides containing the specific modification

  • Include 10-15 amino acids surrounding the modification site

  • Prepare both modified and unmodified peptides for screening

• Antibody Production and Screening Matrix:

• Application Optimization:

  • Determine optimal antibody dilution for each application

  • Establish suitable blocking conditions (typically 5% BSA for phospho-specific antibodies)

  • Validate in multiple experimental systems

This approach yields antibodies that can track specific modifications of At1g68907 during defense responses, providing insight into regulatory mechanisms similar to those observed for other defense-related transcription factors .

How can antibody affinity extraction techniques be combined with mass spectrometry to comprehensively map the At1g68907 interactome during plant defense responses?

Integrating antibody affinity extraction with mass spectrometry creates a powerful platform for interactome analysis:

• Optimized Extraction Protocol:

  • Immobilize At1g68907 antibodies on chromatography support with controlled orientation

  • Condition the column to minimize nonspecific binding while maximizing specific interactions

  • Extract proteins under native conditions to preserve physiologically relevant interactions

• Sequential Elution Strategy:

  • Apply increasing stringency buffers to elute proteins based on interaction strength

  • Collect fractions representing weak, moderate, and strong interactors

  • Include controls for nonspecific binding to the matrix

• Quantitative MS Workflow:

  • Process samples using nano-LC-MS/MS with high-resolution instruments

  • Implement label-free quantification or metabolic labeling for accurate comparison

  • Use specialized software for interaction network construction and visualization

• Validation Pipeline:

  • Confirm key interactions through reciprocal immunoprecipitation

  • Verify functional significance through genetic studies or activity assays

  • Map interaction domains through deletion constructs or peptide arrays

This integrated approach can reveal how the At1g68907 interactome changes during pathogen challenge, potentially identifying regulators similar to how SlSINA3 regulates SlNAC1 stability . The method can also be applied to compare interactome differences between wild-type and immune-compromised plants to identify critical defense-specific interactions .

What are the current limitations of At1g68907 antibody applications in plant defense research?

Current antibody applications face several methodological constraints:

• Specificity Challenges: Distinguishing At1g68907 from closely related NAC family members remains difficult due to conserved domains and epitopes

• Environmental Sensitivity: Plant growth conditions significantly affect protein expression and modification patterns, potentially altering antibody recognition efficiency

• Temporal Resolution: Most current techniques provide snapshots rather than continuous monitoring of dynamic processes during immune responses

• Quantification Accuracy: Standardization across different tissue types and developmental stages is challenging, limiting comparative studies

• Spatial Resolution: Current immunolocalization techniques lack the resolution to distinguish nuclear subcompartments where transcription factors like At1g68907 operate

These limitations parallel challenges faced in other antibody systems, such as those developed for viral proteins like EBV surface proteins , where researchers had to develop multiple antibodies targeting different epitopes to achieve comprehensive coverage.