BZIP28 Antibody
Description
Definition and Target Specificity
The BZIP28 antibody recognizes epitopes on the bZIP28 protein, which contains:
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A single-pass transmembrane domain (TMD) with a helix-breaking glycine residue critical for proteolytic processing .
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A lumenal C-terminal tail with intrinsically disordered regions that bind the ER chaperone BiP .
Most studies employ epitope-tagged versions (e.g., myc-, YFP-, or FLAG-tagged bZIP28) for detection, with antibodies targeting these tags rather than native bZIP28 .
Key Applications in Research
The antibody has been instrumental in:
Subcellular Localization
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Confirming ER membrane localization under unstressed conditions via cell fractionation and protease sensitivity assays .
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Tracking stress-induced relocalization to the Golgi and nucleus using fluorescence microscopy .
Protein-Protein Interaction Studies
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Coimmunoprecipitation (Co-IP) assays demonstrating BiP binding to bZIP28’s lumenal domain under non-stress conditions .
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Identification of interactions with COPII components (e.g., Sar1 GTPase) for ER-to-Golgi transport .
Proteolytic Processing Analysis
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Detecting cleavage products after Site-2 Protease (S2P) activity in the Golgi . Mutations in the TMD (e.g., G329A) block processing, as shown by Western blot .
Critical Research Findings
Key discoveries enabled by BZIP28 antibodies include:
Epitope Tags
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myc/YFP-tagged bZIP28: Used for localization and interaction studies .
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FLAG-tagged BiP isoforms: Co-precipitated with myc-bZIP28 to confirm binding specificity .
Mutant Analysis
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Truncation mutants (e.g., bZIP28Δ355) lacking the BiP-binding lumenal domain show constitutive nuclear localization .
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TMD mutations (e.g., G329A) block proteolytic processing, retained in the Golgi .
Functional Insights
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ER Stress Sensing: BZIP28 monitors ER lumenal conditions via BiP binding. Stress displaces BiP, enabling COPII-mediated transport to the Golgi .
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Transcriptional Regulation: Processed bZIP28 upregulates ER chaperones (e.g., BiP3, calnexin) and heat shock proteins (e.g., HSP70) .
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Thermotolerance: bZIP28 mutants exhibit reduced survival under heat stress, linking its activity to plant resilience .
Limitations and Alternatives
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Most studies rely on overexpression or tagged versions, potentially altering native behavior.
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Native bZIP28 antibodies are rarely described; epitope tags remain the primary detection method.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- These findings reveal that bZIP28 and bZIP60 function independently in plant UPR, and identify an antagonistic role of BI1 in the pro-adaptive signaling mediated by bZIP28, highlighting the complexity of the unfolded protein response (UPR) in plants. PMID: 29124827
- bZIP28 and bZIP60 interact with Ash2 and WDR5a, core components of the COMPASS-like complex. PMID: 25730865
- The lumen-facing C-terminus of bZIP28 plays crucial roles in its movement from the endoplasmic reticulum (ER) to the Golgi, potentially contributing to ER stress sensing. PMID: 23558471
- Data show detectable amounts of YFP-bZIP28 escaping from the ER to nuclei under unstressed conditions in the bip1/bip1 bip2/+ and in the bip1/+ bip2/bip2 and bip3 mutant lines. PMID: 23624714
- bZIP28 is dispersed in the ER in unstressed cells, but its distribution changes upon tunicamycin treatment, preceding its movement into the nuclei via Golgi bodies. PMID: 22335396
- In response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes. PMID: 20207753
- Data indicate that bZIP28 serves as a sensor/transducer in Arabidopsis to mediate ER stress responses related to the unfolded protein response. PMID: 18156219
- The N-terminal fragment of AtbZIP28 translocates to the nucleus in response to ER stress. PMID: 18634751
- bZIP28 is an essential component of a membrane-tethered transcription factor-based signaling pathway that contributes to heat tolerance. PMID: 18849477
Q&A
Experimental Design for Studying BZIP28 Using Antibodies
Q: What experimental approaches can be used to study the role of BZIP28 in endoplasmic reticulum (ER) stress responses using antibodies?
A: To study BZIP28, researchers can employ techniques such as coimmunoprecipitation (co-IP) to examine interactions with other proteins like BiP, and immunofluorescence microscopy to observe BZIP28 localization in the ER and nucleus. Additionally, Western blotting can be used to monitor BZIP28 processing and activation under ER stress conditions .
Antibody Specificity and Validation
Q: How can the specificity of a BZIP28 antibody be validated for research purposes?
A: Validation of antibody specificity involves several steps:
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Western Blotting: Use the antibody to detect BZIP28 in extracts from cells expressing BZIP28 and compare with controls lacking BZIP28.
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Immunoprecipitation: Verify that the antibody specifically pulls down BZIP28 and not other proteins.
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Immunofluorescence: Confirm that the antibody stains the expected cellular compartments (e.g., ER and nucleus) .
Data Interpretation: Contradictions in BZIP28 Localization
Q: How can discrepancies in BZIP28 localization data (e.g., ER vs. nucleus) be resolved?
A: Discrepancies may arise from differences in experimental conditions (e.g., stress levels, cell types). To resolve these, consider:
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Stress Conditions: Ensure consistent ER stress induction methods (e.g., tunicamycin treatment).
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Cellular Context: Use cell fractionation to confirm BZIP28's localization in different cellular compartments .
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Antibody Quality: Verify antibody specificity and sensitivity .
Advanced Techniques for Studying BZIP28 Interactions
Q: What advanced techniques can be used to study interactions between BZIP28 and other proteins like BiP?
A: Techniques such as:
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Protein Cross-Linking: To capture transient interactions between BZIP28 and BiP.
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Phage Display: To identify specific binding sites on BZIP28 for BiP.
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Bimolecular Fluorescence Complementation (BiFC): To visualize interactions in vivo .
Quantitative Analysis of BZIP28 Expression
Q: How can the expression levels of BZIP28 be quantitatively analyzed in response to ER stress?
A: Use quantitative RT-PCR to measure mRNA levels of BZIP28 and its target genes. Additionally, Western blotting can quantify protein levels, and immunofluorescence can assess changes in protein localization and abundance .
Comparative Studies with Other bZIP Factors
Q: How can BZIP28 be compared with other bZIP factors (e.g., bZIP17, bZIP60) in ER stress responses?
A: Compare their:
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Structural Features: Analyze differences in domain structures and potential cleavage sites.
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Expression Patterns: Use qRT-PCR to compare mRNA levels under different stress conditions.
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Functional Roles: Assess their roles in regulating ER stress response genes using knockout mutants or overexpression lines .
Methodological Considerations for BZIP28 Processing
Q: What are key methodological considerations for studying BZIP28 processing under ER stress?
A: Consider:
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Protease Inhibitors: Use inhibitors to prevent unwanted proteolysis during sample preparation.
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Endoglycosidase Treatments: Use EndoH to distinguish between glycosylated and nonglycosylated forms of BZIP28.
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Cell Fractionation: Separate cellular compartments to track BZIP28 movement from ER to Golgi and nucleus .
Network Analysis of BZIP28 and NF-Y Interactions
Q: How can interactions between BZIP28 and NF-Y transcription factors be analyzed in the context of ER stress responses?
A: Use:
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Yeast Three-Hybrid Systems: To identify specific NF-Y subunits interacting with BZIP28.
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Coimmunoprecipitation: To confirm interactions in planta.
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In Vitro Reconstitution: To study transcriptional complex assembly on ERSE-I elements .
ER Stress Induction Protocols
Q: What protocols can be used to induce ER stress for studying BZIP28 activation?
A: Common methods include:
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Tunicamycin (TM) Treatment: Inhibits N-linked glycosylation, causing protein misfolding.
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Dithiothreitol (DTT) Treatment: Disrupts disulfide bonds, leading to protein misfolding.
Data Integration and Modeling
Q: How can data from different experimental approaches be integrated to model BZIP28's role in ER stress responses?
A: Use:
