WIN1 Antibody

What is WIN1 and why is it important in plant research?

WIN1 (WAX INDUCER 1), also known as SHN1 (SHINE 1), is a member of the ERF (ethylene response factor) subfamily B-6 of ERF/AP2 transcription factor family containing one AP2 domain. This transcription factor has gained significant research interest because it regulates cuticle permeability in plants by controlling genes encoding cutin biosynthesis enzymes. WIN1 has been shown to trigger wax production, enhance drought tolerance, and modulate cuticular permeability when overexpressed in Arabidopsis thaliana. Understanding WIN1 function provides fundamental insights into plant adaptation mechanisms to environmental stresses and cuticle formation processes .

What are the known synonyms and identifiers for WIN1?

WIN1 is referenced in scientific literature under several names and identifiers:

• SHN1 (SHINE 1)

• ATSHN1

• WAX INDUCER 1

• Ethylene-responsive transcription factor WIN1

• AT1G15360 (Gene ID)

• Q9XI33 (Protein ID)

The protein is part of a small family that includes two other closely related genes, AT5G25390 (SHN3) and AT5G11190 (SHN2), which exhibit similar phenotypes when overexpressed .

What is the WIN1 antibody used for in plant science research?

The WIN1 antibody is a crucial tool for studying the expression, localization, and function of the WIN1 transcription factor in plant tissues. It enables researchers to:

• Detect the presence and quantity of WIN1 protein through Western blot analysis

• Examine tissue-specific expression patterns through immunohistochemistry

• Investigate protein-protein interactions using co-immunoprecipitation

• Study chromatin binding through chromatin immunoprecipitation (ChIP) assays to identify direct gene targets of WIN1

How should researchers design experiments to study WIN1 function using antibodies?

When designing experiments to study WIN1 function using antibodies, researchers should consider:

• Experimental system selection: Determine whether to use native expression systems or controlled expression systems (e.g., inducible promoters like the DEX-inducible system used with pOp6:WIN1-HA constructs that allowed detection of WIN1-HA protein as early as 90 minutes after induction) .

• Appropriate controls: Include both positive controls (tissues known to express WIN1, like petals where expression is highest) and negative controls (tissues with minimal WIN1 expression or WIN1-silenced lines) .

• Temporal considerations: Plan time-course experiments to capture the sequential activation of target genes, as WIN1 regulates cutin and wax biosynthesis in a two-step process .

• Complementary approaches: Combine antibody-based detection with transcript analysis (RT-PCR) to correlate protein levels with gene expression patterns .

What are the recommended protocols for Western blot analysis using WIN1 antibody?

For optimal Western blot analysis using WIN1 antibody:

• Sample preparation:

  • Extract total protein from plant tissues using a buffer containing protease inhibitors

  • Quantify protein concentration using Bradford or BCA assay

  • Use 20-50 μg of total protein per well

• Electrophoresis and transfer:

  • Separate proteins using 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour

  • Incubate with WIN1 primary antibody (typically 1:1000 dilution) overnight at 4°C

  • Wash 3-4 times with TBST

  • Incubate with appropriate secondary antibody (anti-goat for the polyclonal antibody described in the search results) at 1:5000 dilution for 1 hour

• Detection:

  • Use enhanced chemiluminescence (ECL) detection reagents

  • Expected molecular weight for WIN1 protein should be verified against protein markers

How can researchers validate the specificity of WIN1 antibody?

Validating antibody specificity is crucial for reliable results. Recommended approaches include:

• Genetic validation: Compare antibody signal between wild-type plants and:

  • WIN1 knockout/knockdown lines (e.g., RNAi lines WIN1-R1 and WIN1-R2 that show reduced WIN1 transcript levels to approximately one-third of wild-type)

  • WIN1 overexpression lines (35S:WIN1 or inducible overexpressors)

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before applying to the sample; a specific antibody will show reduced or abolished signal.

• Cross-reactivity assessment: Test the antibody against related proteins (e.g., SHN2 and SHN3) to evaluate potential cross-reactivity, especially since the sequence of the synthetic peptide used for immunization is 93% homologous with sequences in SHN3 and SHN2 .

How can WIN1 antibody be used to identify direct transcriptional targets?

To identify direct transcriptional targets of WIN1:

• ChIP-seq approach:

  • Perform chromatin immunoprecipitation using WIN1 antibody to pull down WIN1-bound DNA fragments

  • Sequence the immunoprecipitated DNA to identify binding sites genome-wide

  • Analyze enriched motifs to define the WIN1 binding consensus sequence

  • This approach has successfully identified LACS2 (long-chain acyl-CoA synthetase) as a likely direct target of WIN1

• ChIP-qPCR validation:

  • Design primers for promoter regions of candidate target genes

  • Perform qPCR on ChIP samples to quantify enrichment relative to input DNA

  • Compare enrichment of targets between wild-type and WIN1-altered expression lines

• Inducible systems analysis:

  • Use DEX-inducible WIN1 expression systems (e.g., pOp6:WIN1-HA) to perform time-course experiments

  • Monitor immediate transcriptional changes after WIN1 induction

  • Distinguish between direct targets (rapidly induced) and indirect targets

How does post-translational modification affect WIN1 antibody detection?

Post-translational modifications (PTMs) can significantly impact antibody detection of WIN1:

• Phosphorylation effects:

  • Phosphorylation of transcription factors often regulates their activity and stability

  • Certain antibodies may have differential affinity for phosphorylated versus non-phosphorylated forms

  • Researchers should consider using phosphatase treatments on a portion of samples to determine if phosphorylation affects detection

• Other PTM considerations:

  • Ubiquitination may affect protein stability and antibody recognition

  • SUMOylation can alter protein localization and function

  • Researchers investigating PTMs should combine WIN1 antibody with specific PTM antibodies in co-immunoprecipitation or Western blot experiments

• Epitope accessibility:

  • Protein-protein interactions or conformational changes may mask antibody epitopes

  • Consider using multiple antibodies targeting different regions of WIN1 if available

What is the relationship between WIN1 and cutin biosynthesis revealed through antibody-based studies?

Antibody-based studies have revealed that WIN1 functions as a master regulator of cutin biosynthesis:

How should researchers address inconsistent WIN1 antibody detection results?

When facing inconsistent WIN1 antibody detection results:

• Sample preparation optimization:

  • Ensure complete protein extraction using appropriate buffers

  • Add protease inhibitors to prevent degradation

  • Consider tissue-specific extraction protocols as WIN1 expression varies by tissue (highest in petals)

• Antibody validation:

  • Verify antibody quality through positive and negative controls

  • Test different antibody dilutions (1:500 to 1:2000) to optimize signal-to-noise ratio

  • Consider lot-to-lot variation in antibody preparations

• Technical considerations:

  • Optimize blocking conditions to reduce background

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure complete membrane washing between antibody applications

• Biological variation awareness:

  • Account for developmental stage differences as WIN1 expression patterns change during development

  • Consider environmental factors that may influence WIN1 expression

  • Evaluate potential redundancy with SHN2/SHN3 that might compensate for WIN1 in certain contexts

How can researchers reconcile contradictory data between WIN1 protein levels and phenotypic observations?

When protein levels detected by WIN1 antibody don't align with expected phenotypes:

• Consider dosage effects:

  • Subtle changes in WIN1 levels may have significant biological effects

  • In RNAi lines, reduction to approximately one-third of wild-type levels was sufficient to alter phenotypes

• Examine tissue-specific effects:

  • Effects of WIN1 modulation are most pronounced in tissues with highest natural expression

  • The impact of WIN1 downregulation was more significant in petals than in leaves

• Evaluate functional redundancy:

  • SHN3 may compensate for reduced WIN1 activity in floral organs

  • Consider analyzing multiple SHINE family members simultaneously

• Analyze downstream targets:

  • Measure expression levels of known targets (e.g., LACS2) to confirm WIN1 activity

  • Conduct cutin and wax composition analyses to verify biochemical phenotypes regardless of protein level observations

What methods exist for quantifying WIN1 protein abundance using antibodies?

For accurate quantification of WIN1 protein:

• Western blot quantification:

  • Use internal loading controls (e.g., actin, tubulin) for normalization

  • Apply densitometry analysis of band intensity using image analysis software

  • Include a standard curve of recombinant protein if absolute quantification is needed

• ELISA-based approaches:

  • Develop sandwich ELISA using WIN1 antibody as capture or detection antibody

  • Compare sample values against a standard curve of recombinant WIN1 protein

• Protein mass spectrometry:

  • Use WIN1 antibody for immunoprecipitation

  • Perform mass spectrometry quantification using labeled reference peptides

  • This approach allows simultaneous identification of interacting proteins

How might WIN1 antibodies enable research on drought stress responses in crops?

WIN1 antibodies could facilitate crop improvement research through:

• Comparative expression studies:

  • Analyze WIN1 expression patterns across crop varieties with different drought tolerance

  • Correlate WIN1 protein levels with cuticle thickness, wax composition, and water retention

  • Identify natural variations in WIN1 that correlate with enhanced stress resilience

• Transgenic crop evaluation:

  • Monitor WIN1 protein levels in engineered crops with altered WIN1 expression

  • Correlate protein abundance with phenotypic outcomes like drought tolerance

  • Assess potential unintended consequences on development or yield

• Environmental response monitoring:

  • Track changes in WIN1 protein levels under various stress conditions

  • Define the temporal dynamics of WIN1 activation in response to drought

  • Determine how WIN1 interacts with other stress response pathways

How can WIN1 antibodies be used to study evolutionary conservation of cuticle regulation?

For evolutionary studies of cuticle regulation:

• Cross-species reactivity assessment:

  • Test WIN1 antibody against orthologs in different plant species across evolutionary distance

  • The antibody shows cross-reactivity with multiple species including Arabidopsis thaliana, Brassica species, Gossypium raimondii, Glycine max, and others

• Comparative regulatory networks:

  • Use immunoprecipitation followed by mass spectrometry to identify interacting partners in different species

  • Compare WIN1-bound promoter elements across species to trace evolution of regulatory networks

• Functional conservation analysis:

  • Correlate antibody-detected protein levels with cuticle composition across species

  • Determine whether the two-step regulation of cutin and wax biosynthesis is evolutionarily conserved

What technologies might enhance WIN1 antibody applications in future research?

Emerging technologies that could enhance WIN1 antibody applications include:

• Single-cell protein analysis:

  • Adapt WIN1 antibodies for use in single-cell proteomics to study cell-specific expression

  • Combine with spatial transcriptomics to correlate protein localization with gene expression patterns

• Microfluidic antibody-based assays:

  • Develop high-throughput platforms for screening WIN1 levels across many samples simultaneously

  • Create lab-on-a-chip approaches for rapid phenotyping of plant lines with altered WIN1 expression

• CRISPR-based genomic tagging:

  • Generate plants with endogenously tagged WIN1 protein compatible with established antibodies

  • Create systems for real-time monitoring of WIN1 dynamics in living plant tissues

• Advanced microscopy techniques:

  • Apply super-resolution microscopy with fluorescently labeled WIN1 antibodies

  • Utilize correlative light and electron microscopy to link WIN1 localization with ultrastructural features of the plant cuticle