DOF2.1 Antibody

What is DOF2.1 and why is it important in plant research?

DOF2.1 (AT2G28510) is a DNA-binding with One Finger (DOF) type transcription factor that functions as a critical component in cytokinin-dependent vascular cell proliferation pathways. It acts downstream of the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex . DOF2.1 specifically controls procambium cell divisions that lead to radial expansion of the root without affecting other cytokinin-regulated processes such as vascular differentiation.

Research has shown that DOF2.1 is induced by cytokinin signaling and likely functions as a direct target of B-type ARR transcription factors (including ARR1, ARR10, and ARR12) . The importance of DOF2.1 lies in its role as a specialized transcriptional regulator that translates cytokinin signals into specific developmental outputs affecting vascular architecture, making it a valuable target for understanding plant vascular development mechanisms.

What experimental applications are suitable for DOF2.1 antibodies?

DOF2.1 antibodies can be employed in multiple experimental applications fundamental to plant developmental biology research:

• Immunolocalization: To detect the spatial distribution of DOF2.1 protein in tissue sections, particularly in root vascular tissues where it's primarily expressed. This approach can complement studies using fluorescent reporter lines (such as pDOF2.1::GUS-GFP) .

• Western blotting: To quantify DOF2.1 protein levels in wild-type versus mutant backgrounds or following hormone treatments, particularly to examine how cytokinin signaling affects DOF2.1 accumulation.

• Chromatin Immunoprecipitation (ChIP): To identify genomic loci directly bound by DOF2.1, helping elucidate its transcriptional targets involved in controlling vascular cell proliferation.

• Co-immunoprecipitation (Co-IP): To investigate potential protein interaction partners of DOF2.1, which might include other transcription factors or components of the cytokinin signaling pathway.

Each application requires specific optimization considering DOF2.1's molecular weight (31.8 kDa) and its tissue-specific expression pattern .

How can you validate the specificity of a DOF2.1 antibody?

Validating DOF2.1 antibody specificity is crucial for ensuring experimental reliability. A comprehensive validation approach should include:

• Genetic controls: Testing the antibody in dof2.1 knockout mutant tissues, which should show significantly reduced or absent signal compared to wild-type. The triple mutant (dof2.1-2 tmo6-1 dof6-2) tissue can also serve as a control, though some signal may persist due to partial redundancy within the DOF family .

• Recombinant protein testing: Using purified recombinant DOF2.1 protein in Western blots to confirm specific binding at the expected molecular weight (31.8 kDa).

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide or purified DOF2.1 protein before application in Western blot or immunostaining to demonstrate that specific binding can be competitively inhibited.

• Cross-reactivity assessment: Testing the antibody against closely related DOF family members like TMO6 and DOF6 to ensure specificity, especially given the functional redundancy observed among these proteins .

• Correlation with reporter lines: Comparing antibody staining patterns with the expression patterns of established pDOF2.1::GUS-GFP reporter lines to confirm consistent localization patterns.

How can DOF2.1 antibodies be used to study the TMO5/LHW-cytokinin signaling pathway?

DOF2.1 antibodies can serve as valuable tools for dissecting the TMO5/LHW-cytokinin signaling pathway through several sophisticated approaches:

• Temporal protein accumulation analysis: Using DOF2.1 antibodies in time-course experiments following TMO5/LHW induction or cytokinin treatment can reveal the kinetics of DOF2.1 protein accumulation relative to other pathway components. This is particularly important considering that DOF2.1 is induced later (3-4 hours) than LOG4 (0.5-1 hour) upon TMO5/LHW induction .

• Spatial co-localization studies: Combining DOF2.1 antibody staining with fluorescent reporters for cytokinin signaling (such as pTCSn::ntdTomato) and auxin response (pDR5revV2::n3GFP) can reveal the precise spatial relationships between hormone signaling domains and DOF2.1 protein localization .

• ChIP-seq following pathway perturbation: Performing DOF2.1 ChIP-seq experiments under conditions where the TMO5/LHW complex is induced or cytokinin signaling is altered can identify condition-specific binding sites and target genes.

• Protein complex dynamics: Using DOF2.1 antibodies for co-IP followed by mass spectrometry in various genetic backgrounds (e.g., arr1 arr10 arr12 triple mutants or wol mutant) can reveal how cytokinin signaling affects DOF2.1's protein interaction network.

• Phosphorylation state analysis: Utilizing phospho-specific DOF2.1 antibodies (if available) to determine whether cytokinin signaling modulates DOF2.1 activity through post-translational modifications.

These approaches collectively can establish DOF2.1's precise position and regulatory mechanisms within the TMO5/LHW-cytokinin pathway governing vascular development.

What are the challenges in detecting DOF2.1 protein in vascular tissues using antibodies?

Detecting DOF2.1 protein in vascular tissues presents several technical challenges that researchers should consider:

• Limited expression domain: DOF2.1 expression is restricted to specific vascular cells, primarily around the phloem poles but notably absent from xylem, centrally located procambium, and phloem lineage cells . This narrow expression domain requires high antibody sensitivity and optimized tissue preparation methods.

• Low abundance: As a transcription factor, DOF2.1 may be expressed at relatively low levels compared to structural or metabolic proteins, necessitating signal amplification techniques or highly sensitive detection methods.

• Tissue penetration barriers: Plant cell walls and vascular tissues can impede antibody penetration. Optimized fixation and permeabilization protocols are essential, potentially including enzymatic cell wall digestion steps.

• Cross-reactivity with related DOF proteins: The DOF transcription factor family includes multiple members expressed in vascular tissues , requiring highly specific antibodies to distinguish DOF2.1 from closely related proteins like TMO6 and DOF6.

• Developmental regulation: DOF2.1 expression is developmentally regulated and influenced by hormonal signaling, meaning detection success may vary depending on developmental stage and physiological conditions of the examined tissues.

To address these challenges, researchers should consider using tyramide signal amplification techniques, optimizing fixation protocols specifically for vascular tissues, and validating results against reporter lines like pDOF2.1::DOF2.1:YFP .

How can ChIP-seq with DOF2.1 antibodies be optimized for identifying downstream targets?

Optimizing ChIP-seq for DOF2.1 requires addressing specific challenges related to plant transcription factor ChIP studies:

• Crosslinking optimization: Standard formaldehyde crosslinking may be insufficient for capturing DOF2.1-DNA interactions. Consider dual crosslinking approaches using DSG (disuccinimidyl glutarate) followed by formaldehyde to stabilize protein-protein interactions before DNA binding.

• Tissue-specific chromatin preparation: Given DOF2.1's vascular-specific expression, enriching for vascular tissues before chromatin preparation will improve signal-to-noise ratio. Consider using fluorescence-activated cell sorting (FACS) with the pDOF2.1::GFP line or rapid vascular bundle isolation techniques.

• Sonication parameters: Optimize sonication conditions specifically for plant vascular tissues, which may have different properties than other plant tissues, to generate chromatin fragments in the ideal 200-500 bp range.

• Antibody selection and validation: Test multiple antibodies raised against different epitopes of DOF2.1, preferably including some recognizing the DNA-binding domain and others targeting unique regions to distinguish from other DOF proteins.

• Controls and normalization: Include appropriate controls such as:

  • Input chromatin control

  • IgG negative control

  • ChIP in dof2.1 knockout background as specificity control

  • Spike-in normalization with a non-plant chromatin source

• Motif analysis integration: Analyze results in conjunction with known DOF-binding motifs (typically containing the core sequence AAAG) to validate binding sites and distinguish direct from indirect targets.

• Integration with gene expression data: Compare ChIP-seq results with transcriptomic changes observed in DOF2.1 overexpression lines and loss-of-function mutants to prioritize functionally relevant targets.

What controls should be included when using DOF2.1 antibodies in developmental studies?

When designing experiments with DOF2.1 antibodies for developmental studies, comprehensive controls are essential:

• Genetic controls:

  • dof2.1 single mutant: Should show reduced signal (though potentially with some compensation from related DOF proteins)

  • dof2.1 tmo6 dof6 triple mutant: Should show significantly reduced signal, though the phenotype is less severe than higher-order mutants of tmo5 or lhw subclades, suggesting additional DOF proteins may be involved

  • DOF2.1 overexpression line (pRPS5A::DOF2.1:GR): Useful as a positive control showing increased signal intensity

• Developmental stage controls:

  • Time-course sampling covering key developmental windows

  • Tissues before and after cytokinin treatment, which should show differential DOF2.1 expression

  • Samples from the wooden leg (wol) mutant background, where cytokinin-dependent induction of DOF2.1 is abolished

• Technical controls:

  • Pre-immune serum application

  • Secondary antibody-only controls

  • Peptide competition assays

  • Positive control tissues with known high DOF2.1 expression (e.g., regions around phloem poles)

• Pathway perturbation controls:

  • Tissues from plants with induced TMO5/LHW expression

  • Samples with exogenous cytokinin treatment

  • B-type ARR mutant backgrounds (arr1, arr10, arr12) where DOF2.1 induction may be affected

Including this comprehensive set of controls will allow for robust interpretation of DOF2.1 antibody signals in developmental contexts.

How can DOF2.1 antibodies be used to investigate redundancy with other DOF-type transcription factors?

Investigating functional redundancy between DOF2.1 and related DOF-type transcription factors is crucial for understanding their collective roles in vascular development. DOF2.1 antibodies can be utilized in the following approaches:

• Comparative immunolocalization: Using DOF2.1-specific antibodies alongside antibodies against TMO6 and DOF6 (the closest homologs) to map their respective expression domains and identify regions of overlap versus unique expression.

• Protein level compensation analysis: Examining protein levels of DOF family members in single, double, and triple mutant backgrounds to detect compensatory upregulation. This approach complements transcript-level studies that have already shown upregulation of TMO6 and DOF6 expression in dof2.1 mutants .

• ChIP-seq comparative analysis: Performing ChIP-seq with antibodies against multiple DOF family members to identify shared versus unique binding sites, revealing potential functional overlap at the target gene level.

• Co-immunoprecipitation studies: Using DOF2.1 antibodies for Co-IP to determine whether DOF2.1 physically interacts with other DOF proteins, potentially forming heterodimers that might explain functional redundancy.

• Sequential ChIP (Re-ChIP): If heterodimer formation is suspected, sequential ChIP with antibodies against different DOF proteins can identify genomic loci bound by multiple DOF factors simultaneously.

• Protein mobility and dynamic analyses: Combining DOF2.1 antibodies with techniques like FRAP (Fluorescence Recovery After Photobleaching) to compare the nuclear mobility and DNA binding dynamics of different DOF proteins in their native contexts.

This multi-faceted approach can provide insights into why the triple dof2.1 tmo6 dof6 mutant shows a less severe phenotype than higher-order mutants of the tmo5 or lhw subclades, suggesting additional DOF family members may contribute to vascular development .

What protein extraction methods are optimal for DOF2.1 detection in plant tissues?

Efficient extraction of DOF2.1 protein from plant tissues requires specialized approaches due to its nature as a nuclear transcription factor and its tissue-specific expression:

Key considerations for all methods:

• Include phosphatase inhibitors to preserve potential phosphorylation states

• Use fresh tissue whenever possible, especially for developmentally regulated processes

• Incorporate DNase treatment when appropriate to release DNA-bound transcription factors

• Consider tissue-specific optimization focusing on vascular-enriched samples

• Test multiple extraction conditions in parallel on the same tissue sample

For all extraction methods, immediate addition of denaturation buffer and boiling is recommended if Western blotting is the downstream application, to minimize proteolytic degradation of low-abundance transcription factors like DOF2.1.

How can immunohistochemistry protocols be optimized for DOF2.1 detection in root cross-sections?

Detecting DOF2.1 in root cross-sections presents unique challenges due to its restricted expression pattern and the nature of plant vascular tissues:

• Tissue Fixation and Embedding:

  • Use a combination of 4% paraformaldehyde with 0.1-0.5% glutaraldehyde for improved protein retention

  • Consider microwave-assisted fixation for better penetration into vascular tissues

  • For embedding, low-melting-point waxes or resin embedding (such as LR White) often provide superior antigen preservation compared to paraffin

• Section Preparation:

  • Optimal section thickness is 4-6 μm for adequate antibody penetration while maintaining tissue integrity

  • Mount sections on adhesive slides (poly-L-lysine or APTES-coated) to prevent loss during processing

  • For cryosections, consider using a cryoprotectant solution optimized for preserving vascular architecture

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective for DOF transcription factors

  • For resin sections, etching with saturated sodium metaperiodate can improve antibody access

  • Consider protease-induced epitope retrieval with a brief proteinase K treatment for formalin-fixed samples

• Blocking and Antibody Incubation:

  • Extended blocking (2-4 hours) with 5% BSA, 5% normal serum, and 0.3% Triton X-100 reduces background

  • Prolonged primary antibody incubation (overnight at 4°C or up to 48 hours) improves signal in dense tissues

  • Include 0.1% Tween-20 and 0.05% Triton X-100 in antibody dilution buffers for enhanced penetration

• Signal Detection and Amplification:

  • Tyramide signal amplification (TSA) can significantly improve detection of low-abundance transcription factors

  • Consider multi-step detection systems using biotin-streptavidin for enhanced sensitivity

  • Quantum dot conjugated secondary antibodies provide excellent signal-to-noise ratio and reduced photobleaching

• Counterstaining and Visualization:

  • Combine DOF2.1 immunodetection with cell wall staining (such as Calcofluor White) for clear delineation of cell types

  • DAPI nuclear counterstaining helps confirm the nuclear localization expected for DOF2.1

  • Consider automated image analysis tools for quantifying signal intensity across different vascular cell types

For optimal results, test the protocol in pRPS5A::DOF2.1:GR inducible lines before and after dexamethasone treatment to establish baseline sensitivity and specificity parameters .

How should researchers interpret DOF2.1 localization data in relation to the cytokinin signaling pathway?

Interpreting DOF2.1 localization data requires careful consideration of its relationship with cytokinin signaling components and developmental contexts:

• Spatial correlation analysis: DOF2.1 localization should be analyzed in relation to cytokinin signaling domains visualized using reporters like pTCSn::ntdTomato. The expression domain of DOF2.1 should partially overlap with, but not be identical to, active cytokinin signaling zones .

• Temporal sequence evaluation: DOF2.1 protein accumulation follows cytokinin pathway activation with a specific delay (3-4 hours after TMO5/LHW induction compared to 0.5-1 hour for LOG4) . This temporal relationship should be evident when comparing DOF2.1 antibody staining with cytokinin reporters at multiple time points after pathway induction.

• Genetic dependency patterns: DOF2.1 localization should show distinctive patterns in different genetic backgrounds:

  • Reduced in wol mutants where cytokinin signaling is compromised

  • Expanded in plants with constitutive B-type ARR activation

  • Altered in specific vascular cell types in tmo5 tmo5-like1 and lhw mutants

• Cell-type specificity interpretation: The notable absence of DOF2.1 from xylem, centrally located procambium, and phloem lineage cells despite active cytokinin signaling in these regions suggests active suppression mechanisms beyond simple cytokinin induction . This pattern is key to understanding DOF2.1's specialized function.

• Functional correlation with phenotypes: DOF2.1 localization should correlate with zones of active vascular cell proliferation but not with zones where cytokinin inhibits differentiation, consistent with DOF2.1's specific role in proliferation without affecting other cytokinin-dependent processes .

When integrating these various data points, researchers should develop models that place DOF2.1 as a specialized effector within the broader cytokinin signaling network, rather than as a general cytokinin response factor.

What approaches can resolve contradictory data from DOF2.1 antibody experiments and reporter lines?

When faced with discrepancies between DOF2.1 antibody detection and reporter line visualization, systematic troubleshooting approaches can help resolve contradictions:

• Epitope masking analysis: Test whether protein interactions or post-translational modifications might be masking the epitope recognized by the antibody under certain conditions. Use multiple antibodies targeting different regions of DOF2.1 to determine if the discrepancy is epitope-specific.

• Reporter construct validation: Examine whether the reporter construct (e.g., pDOF2.1::GUS-GFP) contains all necessary regulatory elements. Some transcription factors have distant enhancers or complex regulatory regions that might be missing in reporter constructs, leading to incomplete expression patterns.

• Protein stability assessment: Investigate whether DOF2.1 protein has different stability in different cell types or conditions. Pulse-chase experiments or treatment with proteasome inhibitors can reveal if post-transcriptional regulation causes differences between transcript (reporter) and protein (antibody) patterns.

• Cross-reactivity resolution: Determine if antibody cross-reactivity with related DOF proteins is causing discrepancies. Perform parallel experiments in single, double, and triple mutant backgrounds (dof2.1, dof2.1 tmo6, dof2.1 tmo6 dof6) to isolate specific signals .

• Technical resolution techniques:

  • Super-resolution microscopy to improve spatial resolution of both signals

  • Flow cytometry of protoplasted reporter lines with antibody staining to quantify co-occurrence at single-cell level

  • Sequential imaging of the same section with direct fluorescence (reporter) followed by immunostaining (antibody)

• Dual validation approach: Generate a translational fusion line where DOF2.1 is tagged with one fluorescent protein (e.g., YFP) and simultaneously use antibodies against a different epitope or tag. Perfect co-localization would validate both approaches, while discrepancies would suggest technical limitations in one method.

The resolution of contradictory data may ultimately reveal important biological insights about DOF2.1 regulation that weren't previously appreciated from either method alone.