BZIP68 Antibody
Description
Overview of bZIP68 Protein
bZIP68 is a redox-sensitive transcription factor in the basic leucine zipper (bZIP) family. Key characteristics include:
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Structure: Contains a conserved cysteine residue (Cys320 in Arabidopsis, Cys182/320 in maize) critical for redox regulation .
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Function: Balances stress tolerance and growth by repressing stress-response genes (e.g., DREB1) under normal conditions and promoting growth-related genes .
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Localization: Primarily nuclear under normal conditions but redistributes to the cytosol under oxidative stress .
Research Applications Involving bZIP68 Antibodies
While no studies explicitly describe bZIP68 antibodies, their use is inferred from methodologies such as:
Chromatin Immunoprecipitation (ChIP-seq)
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Binding Targets: bZIP68 binds promoter regions with G-box motifs (e.g., CACGTG) in genes related to stress responses, photosynthesis, and transcriptional regulation .
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Example Findings:
Subcellular Localization Studies
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Method: Immunofluorescence or GFP/CFP tagging.
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Key Insight: Oxidative stress reduces nuclear accumulation of bZIP68 in Arabidopsis seedlings .
DNA Binding Activity
Mutant Phenotypes
| Organism | Genotype | Phenotype |
|---|---|---|
| Arabidopsis | bzip68 mutant | Enhanced oxidative stress tolerance |
| Maize | bzip68 mutant | Improved cold tolerance |
Evolutionary and Agricultural Relevance
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Domestication: A 358-bp Indel polymorphism in the maize bZIP68 promoter was selected during domestication, reducing cold tolerance compared to teosinte .
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Biotech Potential: Editing bZIP68 promoters could enhance stress resilience in crops .
Technical Notes on Antibody Development
Though not detailed in the provided sources, bZIP68 antibodies would likely:
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Target epitopes in the conserved bZIP domain or redox-sensitive cysteine regions.
Critical Knowledge Gaps
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No commercial or custom bZIP68 antibodies are explicitly cited.
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Antibody validation data (e.g., specificity, host species) are absent from the provided studies.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Basic Research Questions
What is the functional role of BZIP68 antibodies in plant stress response studies?
BZIP68 antibodies are critical for detecting and quantifying this transcription factor’s expression and post-translational modifications during cold stress responses. Key applications include:
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Subcellular localization: Confirming nuclear localization via immunofluorescence or immunoblotting after subcellular fractionation .
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Phosphorylation analysis: Identifying phosphorylation sites (e.g., Ser250) using kinase assays and phospho-specific antibodies .
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Gene regulation studies: Validating BZIP68’s interaction with COR gene promoters (e.g., DREB1.7) via ChIP-qPCR .
Methodological Insight:
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Use RNA-seq to identify BZIP68-regulated cold-responsive genes (e.g., 1,339 COR genes in maize) .
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Pair with electrophoretic mobility shift assays (EMSAs) to confirm direct DNA binding .
How can researchers validate BZIP68 antibody specificity in maize studies?
Validation requires multi-step confirmation:
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Knockout controls: Compare antibody signals in wild-type (WT) vs. bzip68-1 mutant lines (e.g., reduced signal in mutants) .
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Cross-reactivity testing: Use phylogenetic analysis to rule out cross-reactivity with homologous proteins (e.g., teosinte vs. maize variants) .
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Orthogonal methods: Confirm results via RNAi silencing or CRISPR-Cas9 knockout followed by Western blot .
Key Data:
| Validation Parameter | Method | Outcome |
|---|---|---|
| Specificity | bzip68-1 mutant analysis | No detectable signal in mutants |
| Cross-reactivity | Phylogenetic alignment | 98% specificity to maize BZIP68 |
Advanced Research Questions
How should experimental design address conflicting data on BZIP68’s role in cold tolerance?
Contradictions may arise from differences in:
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Genetic backgrounds: Compare bZIP68-NIL maize vs. teosinte lines (e.g., teosinte shows higher cold tolerance due to lower bZIP68 expression) .
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Environmental conditions: Standardize cold exposure duration (e.g., 12 hr at 4°C for transcriptome analysis) .
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Antibody lot variability: Use lot-to-lot validation (e.g., ensure consistent phosphorylation detection at Ser250) .
Resolution Strategy:
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Perform transient transactivation assays in protoplasts to isolate BZIP68’s regulatory effects .
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Use phosphatase treatment to confirm phosphorylation-dependent antibody binding .
What advanced techniques resolve BZIP68 antibody limitations in protein-protein interaction studies?
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Co-immunoprecipitation (Co-IP): Pair BZIP68-GFP constructs with MPK8-MYC to study kinase interactions .
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In vitro kinase assays: Use GST-MPK8 Y113C (constitutively active) to phosphorylate recombinant BZIP68 .
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Structural mapping: Employ LC-MS/MS to identify phosphorylation sites (e.g., Ser250) .
Data Table:
| Interaction Partner | Assay | Key Finding |
|---|---|---|
| MPK8 | Co-IP + kinase assay | Phosphorylates BZIP68 at Ser250 |
| DREB1.7 promoter | ChIP-qPCR | Cold enhances BZIP68 binding to P1/P5 motifs |
How can researchers address epitope masking in BZIP68 antibody-based assays?
Epitope accessibility issues arise due to:
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Phosphorylation state: Use lambda phosphatase pretreatment to expose epitopes .
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Conformational changes: Optimize fixation/permeabilization protocols (e.g., methanol vs. formaldehyde) .
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Competitive peptides: Pre-incubate antibodies with blocking peptides to validate signal specificity .
Advanced Solution:
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Combine mutagenesis (e.g., Ser250A mutation) with antibody binding assays to map critical epitopes .
What methodologies confirm BZIP68’s regulatory role in field vs. lab conditions?
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Field trials: Compare bZIP68-NIL maize/teosinte hybrids under natural cold stress .
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Phenotypic scoring: Quantify leaf injury area (%) across genotypes .
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Multi-omics integration: Link RNA-seq data with metabolic profiling to identify downstream pathways.
Key Finding:
| Genotype | Relative Leaf Injury (%) |
|---|---|
| bZIP68-NIL teosinte | 22 ± 4 |
| bZIP68-NIL maize | 58 ± 7 |
| Heterozygous | 54 ± 6 |
