DOF3.6 Antibody

What is DOF3.6 and what cellular functions does it regulate?

DOF3.6, also known as OBP3 (OCS Binding Factor Binding Protein 3), is a transcription factor belonging to the DOF family. DOF proteins are enzymatically inert adaptor or scaffolding proteins that provide a docking platform for the assembly of multimolecular signaling complexes . Specifically, DOF3.6/OBP3 has been identified as a transcription factor that responds to iron status and triggers iron-deficiency-responsive gene expression in plants .

As a DOF family transcription factor, OBP3 contains a characteristic zinc finger DNA-binding domain with a conserved CX2CX21CX2C motif where four cysteine residues bind metal ions (Zn) . Research demonstrates that DOF3.6/OBP3 directly regulates the expression of bHLH subgroup genes including bHLH38, bHLH39, bHLH100, and bHLH101, which are involved in iron deficiency responses .

What applications are DOF3.6 antibodies suitable for?

Although the search results don't specifically mention applications for DOF3.6 antibodies, antibodies for similar transcription factors like DOK3 are suitable for several common laboratory techniques:

• Western Blotting (WB): For detecting protein expression levels in tissue or cell lysates

• Immunohistochemistry with paraffin-embedded sections (IHC-P): For visualizing protein localization in tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): For examining subcellular localization in cultured cells

When selecting an antibody for DOF3.6/OBP3 research, researchers should verify specificity for their target protein and compatibility with the desired application and species .

How does DOF3.6/OBP3 recognize and bind to DNA?

DOF3.6/OBP3 binds to specific DNA motifs through its DOF domain, which contains one helix and two β-sheets. EMSA (Electrophoretic Mobility Shift Assay) results have demonstrated that the 5-bp [T/A]AAAG motif displays the most effective binding activity with the OBP3 protein . Specifically, AAAAG appears to be more effectively recognized by OBP3 than AAAG, with the position of 3'-G determining the binding affinity of OBP3 DOF domain with DNA in vitro .

The DNA binding mechanism involves:

• Four cysteine residues (C124, C132, C146, and C149) in the conserved CX2CX21CX2C motif that bind zinc ions, stabilizing the interaction between OBP3 DOF domain and DNA

• α-helices of OBP3 DOF domain that embed into the groove of DNA to facilitate binding

• Critical amino acid residues including Y148, W150, R151, Y152, and W153 that are involved in DNA recognition

Mutation studies have confirmed that altering any of the four cysteine residues to alanine eliminates the interaction between OBP3 DOF and DNA, underscoring their critical role in DNA binding .

How should I optimize antibody dilutions for DOF3.6/OBP3 detection in Western blotting?

While specific optimization parameters for DOF3.6/OBP3 antibodies aren't provided in the search results, best practices for similar transcription factor antibodies suggest:

• Begin with manufacturer's recommended dilution range (typically 1/500-1/1000 for primary antibodies in Western blotting)

• Perform a dilution series to determine optimal signal-to-noise ratio

• Include positive controls (tissues/cells known to express DOF3.6/OBP3) and negative controls

• Consider the predicted molecular weight of DOF3.6/OBP3 when analyzing results (for similar proteins like DOK3, the predicted band size is 53 kDa)

For Western blotting applications, secondary antibody selection is also critical. For rabbit-derived primary antibodies, a goat anti-rabbit IgG secondary antibody at approximately 1/50000 dilution has proven effective for similar transcription factor antibodies .

What Design of Experiment (DOE) approach is recommended for optimizing DOF3.6 antibody-based assays?

A Quality by Design (QbD) approach incorporating DOE studies allows for thorough understanding of method design space when establishing antibody-based assays . Though not specific to DOF3.6 antibodies, this methodological framework is applicable:

• Establish an analytical target profile for the antibody-based assay

• Conduct a proof-of-concept study with wide concentration ranges to identify upper/lower asymptotes

• Identify critical method factors using Ishikawa diagrams and cause-effects matrices

• Implement Response Surface Methodology (RSM) DOE studies to optimize key parameters

For antibody-based target binding assays, critical factors often include:

• Antigen coating concentration

• Assay incubation time

• Labeled secondary antibody concentration

Table 1: Example RSM DOE Study Design for Antibody Assay Optimization

This structured approach helps identify optimal assay conditions while understanding factor interactions .

How can ChIP-qPCR be optimized for studying DOF3.6/OBP3 binding to target gene promoters?

Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR) has been successfully used to identify DOF3.6/OBP3 binding to target gene promoters in vivo . Based on published methodologies:

• Generate transgenic constructs expressing tagged DOF3.6/OBP3 (e.g., 35S::OBP3-GFP) to facilitate immunoprecipitation

• Design primers targeting promoter regions of potential target genes (e.g., bHLH38, bHLH39, bHLH100, bHLH101)

• Include G-box containing regions in primer design, as DOF3.6/OBP3 binding shows strong association with G-box elements

• Include controls to verify specificity:

  • Input chromatin samples (pre-immunoprecipitation)

  • Non-specific antibody controls

  • Regions not expected to bind DOF3.6/OBP3

The research demonstrates that ChIP-qPCR can effectively confirm in vivo binding of DOF3.6/OBP3 to multiple bHLH gene promoters, providing crucial validation of protein-DNA interactions identified through other methods .

What approaches are recommended for studying protein-protein interactions involving DOF3.6/OBP3?

DOF3.6/OBP3 is known to interact with other transcription factors like ILR3 (bHLH105), which enhances its binding specificity to DNA motifs . To study such interactions:

• EMSA Supershift Assays: These can detect complex formation between DOF3.6/OBP3 and partner proteins like ILR3 bHLH on DNA fragments. A supershift indicates that proteins can bind to the same DNA fragment simultaneously .

• Mutation Analysis: Generate mutations in:

  • The DOF3.6/OBP3 binding motif ([T/A]AAAG)

  • Partner protein binding sites (e.g., G-box "CACGTG" for ILR3)

  • Observe effects on complex formation and DNA binding

• Genetic Approaches: Compare target gene expression in:

  • Wild-type plants

  • Single mutants (e.g., obp3)

  • Double mutants (e.g., obp3ilr3)

  Research shows that bHLH100 and other bHLH1b subgroup genes have lower expression in obp3ilr3 double mutants compared to obp3 single mutants, confirming functional interaction between these transcription factors .

What structural features of the DOF domain should be considered when designing experiments to study DOF3.6/OBP3 function?

The DOF domain contains critical structural features that determine DNA binding capacity:

• Zinc-Coordinating Cysteines: The four cysteine residues (C124, C132, C146, and C149) in the conserved CX2CX21CX2C motif are essential for metal ion binding and DNA interaction. Mutation of any of these cysteines to alanine eliminates DNA binding .

• α-Helix DNA Recognition Region: Key residues in the α-helix (particularly Y148, W150, R151, Y152, and W153) are involved in DNA recognition. Mutations in R151, Y152, or W153 eliminate DNA-binding activity completely, while mutations in Y148 or W150 reduce binding activity .

• Single Zinc Finger Motif: Unlike many zinc-finger proteins that contain multiple ZF motifs in tandem, DOF proteins including DOF3.6/OBP3 contain only a single ZF motif, which may explain why they often function in complexes with other transcription factors (like ILR3) to enhance binding specificity .

Understanding these structural features is crucial when designing:

• Site-directed mutagenesis experiments

• Truncated protein constructs for functional studies

• Protein-protein interaction studies

• Strategies to disrupt or enhance DNA binding

How can I validate the specificity of a DOF3.6/OBP3 antibody?

Validating antibody specificity is critical for ensuring reliable experimental results. For DOF3.6/OBP3 antibodies, recommended validation approaches include:

• Western Blotting with Known Controls:

  • Lysates from tissues/cells known to express DOF3.6/OBP3

  • Lysates from knockout/knockdown models

  • Recombinant DOF3.6/OBP3 protein as positive control

  • Checking for a single band at the predicted molecular weight

• Immunohistochemistry Controls:

  • Tissues with known expression patterns

  • Blocking peptide competition assays

  • Comparison with RNA expression data (e.g., in situ hybridization)

• Cross-Reactivity Testing:

  • Test against closely related DOF family members (e.g., OBP1, OBP2, OBP4)

  • Arabidopsis contains 37 DOF family members, with OBP3 belonging to a subgroup with four members within a smaller clade

What considerations are important when designing EMSA experiments for DOF3.6/OBP3?

Electrophoretic Mobility Shift Assays (EMSA) have been successfully used to characterize DOF3.6/OBP3 binding to DNA. Key considerations include:

• Protein Preparation:

  • Express DOF3.6/OBP3 DOF domain fused with tags like MBP (maltose-binding protein) for purification

  • Ensure proper folding of the zinc finger domain by maintaining reducing conditions

  • Include zinc in buffers to support the zinc finger structure

• DNA Probe Design:

  • Include the 5-bp [T/A]AAAG motif, with AAAAG showing stronger binding than AAAG

  • Consider the importance of the 3'-G position for binding affinity

  • Include sufficient flanking sequence (at least 5-10 bp on each side)

  • Design mutant probes with 2-bp mutations in the AAAG motif as specificity controls

• Co-Factor Considerations:

  • For studying protein-protein interactions, include purified partner proteins (e.g., ILR3 bHLH)

  • Design probes containing both DOF3.6/OBP3 binding sites and partner protein binding sites (e.g., G-box "CACGTG" for ILR3)

EMSA has successfully demonstrated that OBP3 DOF domain can bind to target DNA sequences in vitro, and that mutations in key residues within the DOF domain eliminate this interaction .

What are common challenges in detecting DOF3.6/OBP3 and how can they be addressed?

While specific challenges for DOF3.6/OBP3 detection aren't detailed in the search results, common issues with transcription factor detection include:

• Low Endogenous Expression Levels:

  • Use enrichment techniques (e.g., nuclear extraction)

  • Employ signal amplification methods

  • Consider transgenic overexpression systems for initial characterization

• Cross-Reactivity with Related Proteins:

  • The DOF family in Arabidopsis contains 37 members with similar DNA-binding domains

  • Use highly specific antibodies raised against unique regions outside the conserved DOF domain

  • Validate specificity against recombinant proteins of closely related family members (OBP1, OBP2, OBP4)

• Nuclear Localization:

  • Ensure nuclear extraction protocols are optimized

  • For immunofluorescence, include nuclear counterstains to confirm localization

  • Consider fixation conditions that preserve nuclear architecture while maintaining epitope accessibility

How should I interpret complex formation between DOF3.6/OBP3 and other transcription factors?

DOF3.6/OBP3 has been demonstrated to interact with other transcription factors such as ILR3 (bHLH105), affecting DNA binding specificity and target gene regulation . When interpreting such interactions:

• EMSA Supershift Patterns:

  • A supershift indicates complex formation on DNA

  • Absence of supershift with mutated binding sites helps identify the requirements for complex formation

  • For DOF3.6/OBP3 and ILR3, research shows that binding of ILR3 bHLH to the G-box is determinant for supershift

• Functional Relevance in vivo:

  • Compare ChIP-qPCR results between wild-type and partner protein mutants

  • Research shows that OBP3 binding to DNA exhibits strong association with G-box motifs

  • Mutation of ILR3 significantly down-regulates OBP3's regulatory ability on target genes

• Target Gene Expression:

  • Expression levels of target genes (e.g., bHLH100) are lower in double mutants (obp3ilr3) compared to single mutants (obp3)

  • This suggests cooperative regulation where both transcription factors contribute to optimal target gene expression

These interpretations help establish a model where DOF3.6/OBP3 and partner proteins like ILR3 function cooperatively to regulate target genes involved in processes such as iron homeostasis .