DPBF2 Antibody

What is DPBF2 and why would researchers need antibodies against it?

DPBF2 (AT3G44460) is a seed-specific transcription factor in Arabidopsis thaliana that plays a critical role in modulating fatty acid composition in developing seeds. It's upregulated by LEAFY COTYLEDON2 (LEC2) and demonstrates transcriptional activity, localizing to the nucleus where it affects the expression of genes involved in fatty acid metabolism. Researchers would need antibodies against DPBF2 to detect, quantify, and study its expression patterns, localization, protein-protein interactions, and binding to DNA target sequences .

How is DPBF2 expressed during seed development?

DPBF2 expression is developmentally regulated during seed formation. Research indicates that DPBF2 is directly regulated by LEC2, which binds to the RY motif (CATGCATGCA) in the DPBF2 promoter region, specifically at positions -36 to -46. When LEC2 was expressed as an effector in experimental conditions, DPBF2 promoter activity increased approximately 130-fold compared to controls. Mutating this RY motif significantly reduced promoter activity, suggesting LEC2 directly controls DPBF2 expression during seed development .

What cellular localization pattern should be expected when using DPBF2 antibodies?

DPBF2 antibodies should primarily detect the protein in the nucleus of plant cells. Experimental evidence using DPBF2-GFP fusion proteins in Arabidopsis protoplasts demonstrated that DPBF2 is targeted to the nucleus, which aligns with its function as a transcription factor. The nuclear localization was confirmed by co-localization studies with a nuclear marker (RFP). This nuclear pattern would be the expected result when using immunofluorescence techniques with DPBF2 antibodies .

How can DPBF2 antibodies be used to study its role in fatty acid biosynthesis regulation?

DPBF2 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to identify direct target genes regulated by DPBF2 in the fatty acid biosynthesis pathway. Research has shown that DPBF2 affects the expression of multiple genes involved in fatty acid metabolism, including FAD2, FAD3, LPCAT1, LPCAT2, PDCT, and FAE1. When DPBF2 was overexpressed, seeds showed altered fatty acid profiles with increased levels of 18:2 and 20:1 fatty acids and decreased levels of 18:1 and 18:3 fatty acids. By using DPBF2 antibodies in ChIP-seq experiments, researchers can map the genome-wide binding sites of DPBF2 and correlate these with transcriptional changes in fatty acid biosynthesis genes .

What are the challenges in detecting DPBF2 protein in dpbf2-1 mutant lines?

When working with dpbf2-1 T-DNA insertion mutants, researchers should be aware that no DPBF2 protein should be detected when using DPBF2 antibodies. The dpbf2-1 mutant has a T-DNA insertion in the second intron of the DPBF2 gene, which prevents proper splicing and results in the absence of DPBF2 expression. RT-PCR and RT-qPCR analyses confirmed that DPBF2 transcript was not detected in developing seeds of dpbf2-1 mutants. Therefore, these mutant lines can serve as excellent negative controls for antibody specificity validation in immunoblotting and immunohistochemistry experiments .

How can researchers investigate DPBF2 interactions with transcriptional complexes using antibodies?

DPBF2 has been shown to form a transcriptional complex with LEC1-LIKE (L1L) and NUCLEAR FACTOR-YC2 (NF-YC2) to regulate genes like PDCT and FAE1. To study these interactions, researchers can use co-immunoprecipitation (Co-IP) assays with DPBF2 antibodies followed by mass spectrometry or immunoblotting for suspected interaction partners. Additionally, proximity ligation assays or fluorescence resonance energy transfer (FRET) techniques using labeled antibodies can visualize these interactions in situ. These approaches would help elucidate how DPBF2 functions within larger transcriptional regulatory networks that control seed fatty acid composition .

What protocols are recommended for optimizing DPBF2 antibody use in immunoprecipitation experiments?

For optimal immunoprecipitation of DPBF2 from plant tissues, researchers should consider tissue-specific extraction protocols that account for DPBF2's nuclear localization. A recommended approach includes:

• Harvest developing seeds at appropriate developmental stages (when DPBF2 expression is highest)

• Use a nuclear extraction buffer containing 50mM HEPES (pH 7.5), 150mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitors

• Sonicate nuclear extracts to shear chromatin if performing ChIP assays

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

• Incubate pre-cleared extracts with DPBF2 antibody (typically 2-5μg) overnight at 4°C

• Capture antibody-antigen complexes with fresh protein A/G beads

• Wash extensively to remove non-specific interactions

This protocol should be optimized based on antibody characteristics and experimental goals .

What controls should be included when validating DPBF2 antibodies for immunostaining applications?

When validating DPBF2 antibodies for immunostaining, the following controls are essential:

• Negative genetic control: Use tissues from dpbf2-1 homozygous mutants which lack DPBF2 expression as confirmed by RT-PCR and RT-qPCR

• Positive control: Use developing seeds from wild-type plants where DPBF2 is known to be expressed

• Dosage control: Include samples from DPBF2/dpbf2-1 heterozygous plants, which should show intermediate staining intensity between wild-type and homozygous mutants

• Overexpression control: Use samples from plants overexpressing DPBF2 (such as those with the 35S-DPBF2 construct), which should show enhanced staining

• Specificity control: Pre-absorb antibody with recombinant DPBF2 protein before staining to confirm signal reduction

• Secondary antibody control: Omit primary antibody to assess non-specific binding of secondary antibody

Research has shown that DPBF2 expression follows a gene dosage effect, with heterozygous plants showing intermediate fatty acid composition phenotypes between wild-type and homozygous mutants, making these excellent controls for antibody validation .

How can DPBF2 antibodies be used to detect conformational changes during transcriptional activation?

To investigate conformational changes in DPBF2 during its activation as a transcription factor, researchers could employ:

• Limited proteolysis coupled with immunoblotting: Compare proteolytic fragments of active versus inactive DPBF2 detected by domain-specific antibodies

• Förster Resonance Energy Transfer (FRET): Use fluorescently labeled antibodies against different DPBF2 domains to detect conformational shifts that alter the distance between epitopes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with immunoprecipitation: Use DPBF2 antibodies to purify the protein from different transcriptional states, then analyze conformational dynamics

• Cross-linking followed by immunoprecipitation: Cross-link protein complexes in vivo, then use DPBF2 antibodies to isolate them for analysis of protein-protein interaction interfaces

These methods would help understand how DPBF2's structure changes when it binds to DNA or interacts with other transcription factors like LEC2, L1L, or NF-YC2 .

How can DPBF2 antibodies be used to study the relationship between DPBF2 and LEC2?

DPBF2 antibodies can be utilized to investigate the regulatory relationship between DPBF2 and LEC2 through:

• Sequential ChIP (ChIP-reChIP): First immunoprecipitate with LEC2 antibodies, then with DPBF2 antibodies to identify genomic regions where both proteins bind

• Protein complex analysis: Use DPBF2 antibodies for co-immunoprecipitation followed by western blotting for LEC2 to determine if they form physical complexes

• Temporal expression analysis: Perform immunohistochemistry with DPBF2 antibodies in wild-type and LEC2 mutant/overexpressor lines to visualize how LEC2 affects DPBF2 expression patterns during seed development

• Promoter binding studies: Combine DPBF2 antibodies with electrophoretic mobility shift assays (EMSAs) to analyze how LEC2 expression affects DPBF2 binding to target gene promoters

Research has shown that LEC2 directly regulates DPBF2 expression by binding to the RY motif in its promoter, increasing promoter activity 130-fold. These techniques would further elucidate this regulatory relationship .

What experimental design is recommended for using DPBF2 antibodies to study fatty acid modification pathways?

A comprehensive experimental design for studying DPBF2's role in fatty acid modification would include:

• Comparative ChIP-seq analysis: Perform ChIP-seq using DPBF2 antibodies in wild-type, dpbf2-1 mutant (negative control), and DPBF2 overexpression lines during seed development stages

• Correlation with transcriptome data: Compare ChIP-seq binding sites with RNA-seq data to identify direct DPBF2 targets among fatty acid modification genes

• Validation of binding sites: Use DPBF2 antibodies in ChIP-qPCR to confirm binding to promoters of key genes (FAD2, FAD3, LPCAT1, LPCAT2, PDCT, and FAE1)

• Functional assays: Combine immunoprecipitation with activity assays to determine if DPBF2 binding correlates with changes in enzyme activities

• Co-occupancy studies: Use sequential ChIP to determine if DPBF2 co-occupies promoters with other transcription factors involved in fatty acid synthesis

This approach would provide a comprehensive understanding of how DPBF2 directly regulates genes involved in fatty acid desaturation, elongation, and acyl-editing processes, explaining the altered fatty acid profiles observed in DPBF2 mutant and overexpressor lines .

What factors might affect DPBF2 antibody detection in different developmental stages of seeds?

Several factors can influence DPBF2 antibody detection across seed development stages:

• Expression timing: DPBF2 is upregulated by LEC2 during specific developmental windows; sampling outside these periods may result in weak or absent signals

• Protein modifications: Post-translational modifications might mask epitopes at certain developmental stages

• Protein complex formation: DPBF2 interactions with other proteins (such as L1L and NF-YC2) might affect antibody accessibility

• Fixation sensitivity: Some developmental stages may require different fixation protocols to preserve DPBF2 epitopes

• Tissue penetration issues: As seeds develop and accumulate storage compounds, antibody penetration may become more challenging

Researchers should optimize protocols based on the specific developmental stage being studied, potentially using different extraction methods or epitope retrieval techniques for mature versus developing seeds .

How should researchers interpret contradictory results between transcript levels and protein detection when using DPBF2 antibodies?

When facing discrepancies between DPBF2 transcript levels and protein detection:

• Post-transcriptional regulation: Consider microRNA-mediated regulation or RNA stability factors that might cause transcript presence without protein translation

• Protein stability: Investigate if DPBF2 undergoes rapid turnover in certain conditions despite transcript presence

• Technical considerations: Verify antibody specificity using dpbf2-1 mutant controls and check extraction protocols for compatibility with the specific tissue

• Epitope masking: Examine if post-translational modifications or protein-protein interactions might be masking the epitope

• Subcellular compartmentalization: Ensure extraction methods effectively isolate the nuclear fraction where DPBF2 is localized

Research has shown that DPBF2 has transcriptional activity and localizes to the nucleus, so protocols should be optimized to effectively extract and detect nuclear proteins. Comparisons between wild-type and genetically modified lines (heterozygous, homozygous mutant, and overexpression) can help validate detection methods .