DOF5.1 Antibody

What is DOF5.1 and why is it significant in plant research?

DOF5.1 (AtDof5.1 in Arabidopsis) belongs to the plant-specific Dof transcription factor family characterized by a highly conserved DNA-binding domain. This transcription factor is particularly important in regulating leaf axial patterning and has been shown to interact directly with HD-ZIP transcription factors . DOF5.1 is predominantly expressed in vascular tissues and plays significant roles in developmental processes. Understanding DOF5.1 function provides insights into plant developmental regulation and signaling pathways, making it a valuable target for agricultural biotechnology research.

What applications are most suitable for DOF5.1 antibodies?

DOF5.1 antibodies are primarily used in applications including Western blotting (WB), immunohistochemistry (IHC), chromatin immunoprecipitation (ChIP), and enzyme-linked immunosorbent assay (ELISA). Based on validation approaches for other plant transcription factor antibodies, researchers should expect optimal performance in Western blot applications at concentrations between 0.1-0.5 μg/ml . For immunolocalization studies, higher concentrations may be required depending on tissue fixation methods. Verification of antibody specificity should be conducted with positive controls such as Arabidopsis vascular tissues where DOF5.1 is known to be expressed.

How do researchers confirm specificity when working with DOF5.1 antibodies?

Confirming specificity requires multiple validation approaches:

• Molecular weight verification: DOF5.1 should appear at its predicted molecular weight

• Positive and negative tissue controls: Compare expression in vascular tissues (positive) versus non-vascular tissues (negative)

• Knockout/knockdown validation: Test antibodies in dof5.1 mutant lines

• Blocking peptide experiments: Pre-incubation with immunizing peptide should eliminate specific signal

• Cross-reactivity assessment: Test against recombinant proteins of closely related DOF family members

Proper validation prevents experimental artifacts and ensures reliable data interpretation in downstream applications.

What is the optimal protein extraction protocol for DOF5.1 detection in plant tissues?

For effective DOF5.1 detection in plant tissues, we recommend the following extraction protocol:

When extracting nuclear proteins like DOF5.1, it's critical to include phosphatase inhibitors if studying post-translational modifications and to work quickly to minimize protein degradation. This extraction method has been optimized based on protocols for similar plant transcription factors.

How should immunohistochemistry protocols be optimized for DOF5.1 detection in plant tissues?

Optimizing immunohistochemistry for DOF5.1 requires special considerations due to plant cell walls and tissue-specific expression patterns:

• Fixation: Use 4% paraformaldehyde with vacuum infiltration; avoid over-fixation which can mask epitopes

• Antigen retrieval: Critical step - use citrate buffer (pH 6.0) with heat treatment to expose masked epitopes

• Cell wall digestion: Partial enzymatic digestion with cellulase/pectinase improves antibody penetration

• Antibody concentration: Start with 1:100-1:500 dilution and optimize through titration

• Incubation conditions: Extend primary antibody incubation to 16-24 hours at 4°C for better penetration

• Detection system: Use high-sensitivity detection systems (TSA amplification when necessary)

• Controls: Include tissue from dof5.1 mutant plants as negative control

Each plant species and tissue type may require further protocol adjustments. Compare transcriptional and translational patterns as DOF family members may exhibit cell-to-cell movement .

What are the key considerations for using DOF5.1 antibodies in ChIP experiments?

When performing ChIP with DOF5.1 antibodies, consider these critical factors:

• Crosslinking optimization: Use 1% formaldehyde for precisely 10 minutes; over-crosslinking can reduce efficiency

• Chromatin fragmentation: Aim for 200-500bp fragments; optimize sonication cycles for plant tissues

• Antibody validation: Verify that the antibody immunoprecipitates DOF5.1 efficiently before ChIP experiments

• Input normalization: Essential for quantitative analysis of binding sites

• Negative controls: Include IgG controls and non-target regions lacking DOF binding motifs

• Positive controls: Include genomic regions containing the DOF consensus sequence (AAAG/CTTT)

• Sequential ChIP: For studying complexes with other transcription factors mentioned in literature

DOF5.1 is known to interact with other transcription factors, so sequential ChIP may reveal important co-regulatory mechanisms. Target validation should include analysis of DOF binding motifs in identified regions.

How can DOF5.1 antibodies be used to study intercellular protein trafficking?

Several DOF family transcription factors have been identified as mobile proteins that can move between cells, including AtDof4.1 and potentially seven other Dof TFs (AtDof1.1, AtDof2.1, AtDof2.2, AtDof2.4, AtDof3.2/DOF6, AtDof3.6/OBP3, AtDof3.7/DAG1) . To investigate whether DOF5.1 exhibits intercellular movement:

• Immunolocalization with high resolution: Compare protein localization with transcriptional domains using tissue-specific reporters

• Microinjection studies: Labeled antibodies can track protein movement in real-time

• Cell-specific expression systems: Express DOF5.1 under cell-specific promoters and detect protein in adjacent cells

• Grafting experiments: Use species-specific antibodies to detect movement across graft junctions

• FRAP analysis: With fluorescently-tagged DOF5.1 to measure mobility rates

Critical controls should include comparison with known mobile (AtDof4.1) and non-mobile transcription factors. Look for the intercellular trafficking motif (ITM) that includes the zinc finger motif and nuclear localization signal, which has been shown to be required for movement in other DOF family members .

What approaches help distinguish between DOF5.1 and other DOF family members?

Distinguishing between closely related DOF family members requires strategic approaches:

Since the DOF domain is highly conserved, epitopes for antibody generation should target variable regions outside this domain. Validation in knockout/knockdown lines for each family member provides the most definitive confirmation of specificity.

How can researchers utilize DOF5.1 antibodies to investigate its role in light-responsive pathways?

DOF transcription factors have been implicated in light signaling pathways, with several members (including AtDof3.7/DAG1, AtDof2.5/DAG2, AtDof1.5/COG1, and AtDof3.6/OBP3) shown to regulate photomorphogenesis processes like germination and hypocotyl growth . To investigate DOF5.1's potential role in light response:

• Light/dark differential expression: Compare DOF5.1 protein levels in light vs. dark-grown seedlings using quantitative Western blotting

• Chromatin dynamics: Use ChIP-seq to identify light-dependent changes in DOF5.1 genomic binding

• Protein interaction studies: Co-IP with known light signaling components (phytochromes, cryptochromes)

• Post-translational modifications: Phospho-specific antibodies to detect light-dependent modifications

• Circadian regulation: Time-course sampling to detect rhythmic accumulation patterns

Research should examine interactions with established light signaling pathways, particularly phytochrome and cryptochrome signaling which are known to involve other DOF family members .

How should researchers address non-specific binding when using DOF5.1 antibodies?

Non-specific binding is a common challenge with plant transcription factor antibodies. Address these issues systematically:

• Increase blocking stringency: Use 5% BSA or milk with 0.1-0.3% Triton X-100

• Optimize antibody concentration: Titrate to find minimal concentration that produces specific signal

• Increase washing stringency: Add moderate salt (150-300mM NaCl) to wash buffers

• Pre-absorb antibody: Incubate with protein extract from knockout plants before use

• Modify fixation protocols: Over-fixation can increase background in immunohistochemistry

• Validate with alternate antibodies: Use antibodies targeting different epitopes of DOF5.1

• Counter-screen: Test reactivity against recombinant DOF family proteins

When evaluating Western blot results, always look for a single band at the expected molecular weight (similar to the calculated 23kDa observed for other transcription factors) . Multiple bands may indicate degradation, post-translational modifications, or non-specific binding.

How can researchers interpret discrepancies between mRNA expression and protein localization data?

Discrepancies between transcriptional and translational patterns are particularly relevant for DOF transcription factors, as several family members have been shown to move between cells. For instance, AtDof4.1 shows expansion from pericycle to endodermis, while AtDof3.7/DAG1 shows different localization between transcriptional and translational fusions .

When encountering such discrepancies with DOF5.1:

• Verify antibody specificity: Confirm antibody recognizes only DOF5.1

• Compare methods: Use multiple detection methods (antibodies, fluorescent protein fusions)

• Examine time dynamics: Temporal differences between transcription and translation

• Consider protein movement: Investigate intercellular trafficking like that observed in AtDof4.1

• Assess mRNA movement: Some transcription factors show mobile mRNA

• Evaluate post-transcriptional regulation: Examine protein stability and turnover rates

The expansion of protein localization beyond the transcriptional domain strongly suggests protein movement, as has been demonstrated for some DOF family members that contain intercellular trafficking motifs (ITM) .

What controls are essential when validating new lots of DOF5.1 antibodies?

Consistent antibody performance across lots is critical for research reproducibility. Essential validation controls include:

Document all validation results, including images of Western blots showing full molecular weight ranges and quantitative comparisons between lots. This approach mirrors the comprehensive validation employed for other antibodies in research settings .

How can DOF5.1 antibodies be integrated with proteomics approaches to study transcription factor complexes?

Modern proteomics offers powerful approaches to study DOF5.1-containing complexes:

• IP-MS workflows: Immunoprecipitate DOF5.1 followed by mass spectrometry to identify interacting partners

• Proximity labeling: BioID or APEX2 fusions to DOF5.1 to identify proximal proteins in vivo

• Cross-linking MS: To capture transient interactions within transcriptional complexes

• Targeted proteomics: MRM/PRM assays for quantitative analysis of DOF5.1 and interactors

• PTM mapping: Phosphorylation, SUMOylation, and ubiquitination analysis

These approaches can reveal interactions with other transcription factor families known to interact with DOF members, such as bHLH and HD-ZIP factors . Special consideration should be given to tissue-specific complex formation, particularly in vascular tissues where DOF5.1 is predominantly expressed.

What considerations apply when developing degrader antibody conjugates (DACs) targeting DOF5.1?

While primarily a research concept, developing DACs for plant transcription factors like DOF5.1 would require specialized approaches:

• Target validation: Confirm DOF5.1 accessibility in plant cell nuclei

• Antibody selection: Choose antibodies with high specificity and internalization capacity

• Linker design: Optimize for stability in plant cellular environments

• Degrader selection: Select plant-compatible degraders that engage plant proteasome systems

• Validation assays: Develop plant cell-based assays measuring target degradation

The DAC approach would combine antibody specificity with targeted protein degradation, potentially allowing temporal control over DOF5.1 function . This represents an emerging frontier in plant molecular biology research that could complement genetic approaches.

How can CRISPR-based approaches complement antibody studies of DOF5.1?

Integrating CRISPR technologies with antibody-based approaches creates powerful research systems:

• Endogenous tagging: CRISPR knock-in of epitope tags for improved antibody detection

• Validation systems: Generate clean knockout lines for antibody validation

• Domain mutation: Create specific functional domain mutants to map antibody epitopes

• Inducible systems: Combine with antibody detection for temporal studies

• Single-cell analysis: CRISPR screens with antibody-based readouts

For example, CRISPR-mediated tagging of DOF5.1 with a small epitope tag allows antibody detection while maintaining native regulation and expression levels. This approach is particularly valuable for studying developmental processes where DOF5.1 interacts with HD-ZIP transcription factors to regulate leaf patterning .