BZIP11 Antibody

What is BZIP11 and why is it important in plant research?

BZIP11 (also known as ATB2, ATBZIP11, or G-BOX BINDING FACTOR 6/GBF6) is a transcription factor belonging to the basic leucine-zipper (bZIP) family in Arabidopsis thaliana. It serves as a crucial transcriptional regulator involved in plant sugar and amino acid metabolism . Research indicates that BZIP11 acts as a susceptibility factor during Pseudomonas syringae infection, influencing the transcription of multiple nutrient transporters that facilitate nutrient efflux from secretory cells . Its importance extends to auxin-mediated primary root growth regulation , making it a significant protein for understanding plant development and pathogen responses.

What are the available types of BZIP11 antibodies for research?

Researchers typically have access to several types of BZIP11 antibodies:

• Polyclonal antibodies: Recognize multiple epitopes on the BZIP11 protein, providing robust detection but potentially lower specificity

• Monoclonal antibodies: Target specific epitopes, offering higher specificity but potentially lower sensitivity

• Recombinant antibodies: Produced using recombinant DNA technology for consistent performance

• Phospho-specific antibodies: Detect phosphorylated forms of BZIP11, particularly relevant since its dimerization and activity depend on phosphorylation status

How is BZIP11 structurally and functionally related to other bZIP transcription factors?

BZIP11 belongs to Group S1 of the bZIP transcription factor family and can form both homodimers and heterodimers with Group C bZIPs . The dimerization patterns significantly influence DNA binding preferences and target gene regulation. According to DAP-seq and dDAP-seq studies, BZIP11 can bind DNA as a homodimer and forms functional heterodimers with bZIP9, altering DNA binding specificity . This heterodimer formation expands its regulatory repertoire, enabling control of diverse biological functions including responses to auxin, jasmonic acid, and salicylic acid .

What are the optimal conditions for using BZIP11 antibodies in ChIP experiments?

For Chromatin Immunoprecipitation (ChIP) using BZIP11 antibodies:

• Crosslinking protocol: Fix plant tissue with 1% formaldehyde for 10 minutes at room temperature followed by quenching with 0.125M glycine.

• Sonication parameters: Optimize to achieve DNA fragments of 200-500bp.

• Antibody concentration: Use 2-5 μg of BZIP11-specific antibody per ChIP reaction.

• Positive control regions: Include the IAA3 promoter which has been verified as a direct binding target of BZIP11 .

• Negative controls: Include ACTIN7 promoter regions and IAA3 coding/3'UTR regions which show minimal enrichment .

Research by Prior et al. demonstrated successful ChIP using root material from XVE-bZIP11 plants, showing strong enrichment of IAA3 promoter fragments compared to wild-type controls .

How can BZIP11 antibodies be validated for specificity and sensitivity?

A multi-step validation approach is recommended:

It's critical to validate antibody performance in the specific experimental context, particularly when studying interactions with other bZIP family members due to potential cross-reactivity with homologous regions.

What methodologies are effective for studying BZIP11 phosphorylation states?

BZIP11 activity is regulated by phosphorylation, making phosphorylation-state analysis important. Effective methodologies include:

• Phospho-specific antibodies: Use antibodies specifically recognizing phosphorylated residues of BZIP11.

• Phos-tag SDS-PAGE: Incorporate Phos-tag molecules in gels to retard phosphorylated protein migration, allowing separation of different phosphorylation states.

• In-gel kinase assays: As demonstrated in studies with related bZIP63, immobilize BZIP11 in gels and detect kinase activity towards it .

• Mass spectrometry: Use LC-MS/MS to identify phosphorylation sites after trypsin digestion of immunoprecipitated BZIP11.

• Protein kinase identification: Apply the approach used for bZIP63, where kinases were identified by excising bands corresponding to signals from in-gel kinase assays followed by LC-MS/MS analysis .

How can BZIP11 antibodies be used to investigate dimerization with other bZIP transcription factors?

To investigate BZIP11 dimerization:

• Co-immunoprecipitation (Co-IP): Use BZIP11 antibodies to pull down protein complexes from plant extracts, followed by western blotting with antibodies against potential partner proteins (bZIP9, bZIP10, bZIP25, bZIP63).

• Double ChIP-seq (sequential ChIP): First immunoprecipitate with BZIP11 antibodies, then perform a second immunoprecipitation with antibodies against potential dimerization partners to identify genomic regions bound by specific heterodimers.

• Proximity ligation assay (PLA): Detect protein-protein interactions in situ using BZIP11 antibodies paired with antibodies against potential dimerization partners.

Recent research using dDAP-seq (double DNA affinity purification sequencing) has revealed that BZIP11 forms heterodimers with Group C bZIPs (particularly bZIP9), altering DNA binding preferences and expanding the regulatory repertoire . This approach could be complemented with antibody-based techniques for in vivo validation.

What approaches can resolve the dual role of BZIP11 in immunity and susceptibility to pathogens?

BZIP11 presents an interesting paradox as both a susceptibility factor and a potential immunity regulator . To investigate this dual role:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq): Use BZIP11 antibodies to identify direct binding targets during different phases of pathogen infection.

• Temporal analysis: Apply antibody-based techniques at different timepoints during infection to track BZIP11 activity and association with different protein complexes.

• Cell-type specific analysis: Employ fluorescence-activated cell sorting (FACS) followed by immunoprecipitation to study BZIP11 function in different cell types during infection.

• Genetic complementation: In bzip11 knockdown lines showing reduced pathogen susceptibility, perform domain-specific complementation to separate functions related to immunity repression versus nutrient transport activation.

Research by Prior et al. indicates that BZIP11 may function as both a repressor of immunity and an activator of nutrient pathways for pathogens , making it a complex regulatory node requiring sophisticated experimental approaches to fully characterize.

How can researchers distinguish between direct and indirect transcriptional targets of BZIP11 using antibody-based approaches?

Distinguishing direct from indirect targets requires integrated approaches:

• ChIP-seq with BZIP11 antibodies: Identify genome-wide binding sites to establish potential direct targets.

• Integration with RNA-seq data: Correlate binding events with transcriptional changes in bZIP11 overexpression or knockdown lines.

• TIME-ChIP (Temporally resolved ChIP): Study the kinetics of BZIP11 binding following induction.

• TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors): Compare with previous results for related bZIP1 which identified three classes of targets: poised (Class I), stable (Class II), and transient (Class III) .

• Motif analysis: Correlate binding events with the presence of known BZIP11 binding motifs (primarily ACGT-containing elements).

Research indicates that BZIP11 targets include UmamiT transporters and genes involved in amino acid metabolism , but differentiating direct from indirect regulation requires temporal resolution of binding events and transcriptional responses.

How can researchers overcome non-specific binding when using BZIP11 antibodies?

Non-specific binding is a common challenge with transcription factor antibodies. To overcome this:

• Optimization of blocking conditions: Test different blocking agents (BSA, milk, normal serum) at varying concentrations (3-5%).

• Antibody titration: Determine the minimum effective concentration to reduce background.

• Pre-adsorption: Incubate antibodies with lysates from bZIP11 knockdown plants (bzip11-s1, bzip11-s2) to remove non-specific antibodies.

• Stringent washing: Increase washing steps or modify buffer composition after immunoprecipitation.

• Alternative epitopes: If targeting the highly conserved bZIP domain causes cross-reactivity, use antibodies recognizing unique regions of BZIP11.

The high sequence homology between BZIP11 and closely related factors has previously hampered knockdown approaches using artificial microRNA techniques , suggesting similar challenges may exist for antibody specificity.

What are the key considerations when interpreting ChIP-seq data generated using BZIP11 antibodies?

When interpreting BZIP11 ChIP-seq data:

• Dimerization complexity: Consider that BZIP11 can form both homodimers and heterodimers with different binding preferences. ChIP-seq will capture the composite of all BZIP11-containing complexes.

• Motif analysis: Expect enrichment of ACGT-containing elements, but be aware that heterodimers with Group C bZIPs may show altered motif preferences .

• Integration with transcriptome data: Compare binding sites with expression changes in bZIP11 overexpression or knockdown lines to identify functional binding events.

• Biological context: Consider the specific conditions (energy status, pathogen infection) as BZIP11 function is context-dependent.

• Resolution limitations: Be aware that ChIP typically has a resolution of approximately 1000bp, which may lead to some enrichment of adjacent regions .

How can experimental artifacts be distinguished from genuine BZIP11 binding events?

To distinguish artifacts from genuine binding:

• Use multiple antibodies: Compare results from different antibodies recognizing distinct epitopes of BZIP11.

• Include appropriate controls: Perform ChIP in bZIP11 knockdown lines (bzip11-s1, bzip11-s2) to identify background signal.

• Validate with orthogonal methods: Confirm key findings with techniques like ChIP-qPCR, EMSA, or reporter gene assays.

• Compare with published DAP-seq data: Cross-reference with available direct DNA-binding data for BZIP11 homodimers and heterodimers .

• Biological replicates: Ensure reproducibility across independent experiments and biological samples.

Research has shown that BZIP11 directly binds the IAA3 promoter but shows minimal enrichment of ACTIN7 promoter or IAA3 coding/3'UTR regions , providing positive and negative control regions for validation.

How should researchers analyze changes in BZIP11 levels during pathogen infection?

When analyzing BZIP11 levels during infection:

• Time-course analysis: Track BZIP11 protein levels at multiple timepoints following pathogen inoculation using quantitative western blotting with BZIP11 antibodies.

• Spatial analysis: Combine with tissue-specific extraction or immunohistochemistry to determine if BZIP11 induction is localized to infection sites.

• Post-translational modifications: Assess phosphorylation status changes, as this affects BZIP11 activity and dimerization .

• Correlation with pathogen growth: Compare BZIP11 induction kinetics with bacterial population dynamics, as overexpression of BZIP11 leads to increased bacterial growth .

• Target gene expression: Monitor expression of confirmed BZIP11 targets like UmamiT transporters as functional readouts of BZIP11 activity .

Research has identified BZIP11 as a susceptibility factor during Pseudomonas syringae infection, with knockdown lines showing reduced bacterial growth , making its expression pattern during infection particularly relevant.

What insights can BZIP11 antibodies provide regarding energy signaling and transcriptional reprogramming?

BZIP11 antibodies can reveal:

• Energy-dependent localization: Track BZIP11 subcellular localization changes in response to energy status using immunofluorescence or cell fractionation followed by western blotting.

• Protein complex formation: Identify energy-dependent interaction partners through co-immunoprecipitation followed by mass spectrometry under different energy conditions.

• Target binding dynamics: Apply ChIP-seq under normal versus energy-depleted conditions to map changes in genomic binding sites.

• Phosphorylation status: Monitor energy-dependent post-translational modifications using phospho-specific antibodies or general BZIP11 antibodies combined with Phos-tag gels.

Research shows that BZIP11 is a low-energy activated transcription factor that links energy signaling to auxin-mediated root growth , suggesting its activity and interactions change significantly with cellular energy status.

How can researchers incorporate BZIP11 antibody data with other omics approaches to build comprehensive regulatory networks?

To build comprehensive regulatory networks:

• Integration with transcriptomics: Correlate BZIP11 binding sites (ChIP-seq) with differential expression in bZIP11 mutants to establish direct versus indirect targets.

• Metabolomics correlation: Link BZIP11 activity (measured by antibody-based methods) with metabolite profiles, particularly amino acids and sugars that are known to be affected by BZIP11 .

• Protein interaction networks: Combine co-immunoprecipitation data with known protein-protein interaction networks to place BZIP11 in broader regulatory contexts.

• Multi-omics data integration: Develop computational models incorporating ChIP-seq, RNA-seq, and metabolomics data to predict BZIP11's role in various conditions.

• Network visualization: Use tools like Cytoscape to visualize and analyze BZIP11-centered regulatory networks incorporating antibody-derived data.

Research has shown that BZIP11 target genes are enriched for specific GO terms related to responses to auxin, jasmonic acid, salicylic acid, water, and hypoxia , providing a foundation for building comprehensive regulatory networks.