UGT89C1 Antibody

What is UGT89C1 and why is it important to study with antibodies?

UGT89C1 (UDP-glycosyltransferase 89C1) is a plant enzyme from Arabidopsis thaliana that functions as a rhamnosyltransferase, catalyzing the transfer of rhamnose from UDP-β-l-rhamnose to specific acceptor molecules, particularly flavonoids such as quercetin. This enzyme plays a critical role in plant secondary metabolism by enhancing the bioactivity and bioavailability of natural products. Antibodies against UGT89C1 are valuable tools for tracking protein expression, localization, and studying the role of this enzyme in flavonoid metabolism and plant development. The enzyme's importance in glycosylation pathways makes it a relevant target for investigating plant responses to various stresses and developmental conditions.

What epitopes should be targeted when designing a UGT89C1 antibody?

When designing antibodies against UGT89C1, several strategic epitopes should be considered:

• Catalytic residues: His21 is essential for nucleophilic attack during glycosylation and could serve as a distinctive epitope.

• Donor specificity residues: Asp356, His357, Pro147, and Ile148 are key residues for sugar donor recognition and specificity for UDP-β-l-rhamnose .

• Non-conserved regions: Target unique regions that distinguish UGT89C1 from other UGTs to avoid cross-reactivity.

• N-terminal regions: For recombinant proteins, the 10xHis-tag at the N-terminus could be targeted for detection purposes .

When using recombinant UGT89C1 as an immunogen, note that the protein is typically expressed in E. coli with an N-terminal 10xHis-tag, with a molecular weight of 51.6 kDa and high purity (>85% by SDS-PAGE) .

What immunolocalization protocols work effectively for UGT89C1 detection?

For effective immunolocalization of UGT89C1, researchers should consider the following protocol components:

• Fixation: Use 4% paraformaldehyde to preserve cellular architecture while maintaining antigen accessibility .

• Automated approach: Utilize an InsituPro VSi pipetting robot for consistent whole-mount protein immunolocalization, which can reduce experimental variability .

• Primary antibody dilution: The optimal dilution may vary by antibody source, but related plant proteins have been successfully detected with antibody dilutions of 1:800 .

• Secondary antibody selection: Use fluorescently-labeled secondary antibodies like Cy3 anti-rabbit at 1:600 dilution for visualization .

• Blind analysis procedure: To avoid bias in localization studies, implement a numbering system for samples and evaluate microscope slides without knowing their identities until after analysis is complete .

This approach has been successfully used for PIN protein localization in plant tissues and can be adapted for UGT89C1 studies with appropriate antibody selection.

How can recombinant UGT89C1 be produced for antibody generation?

For antibody production, high-quality recombinant UGT89C1 protein can be generated following these methodological steps:

• Expression system: Use E. coli BL1 (DE3) cells, which have been successfully employed for expressing plant UGTs .

• Vector design: Incorporate an N-terminal tag (10xHis or GST) to facilitate purification and potentially enhance immunogenicity .

• Verification: Confirm recombinant plasmids via Sanger DNA sequencing before protein expression .

• Purification method: Employ affinity chromatography using columns suitable for the chosen tag .

• Protein quantification: Use Bradford assay for precise determination of protein concentration .

• Quality control: Verify protein purity and size (51.6 kDa) using SDS-PAGE before immunization .

• Buffer optimization: Prepare the purified protein in Tris/PBS-based buffer with glycerol (5-50%) for liquid formulations or 6% Trehalose for lyophilized preparations.

This approach ensures production of highly pure recombinant UGT89C1 suitable for generating specific antibodies.

How can UGT89C1 antibodies be used to investigate flavonoid metabolism in plants?

UGT89C1 antibodies offer powerful tools for investigating flavonoid metabolism through several sophisticated approaches:

• Co-localization studies: Combine UGT89C1 antibodies with flavonoid-specific stains to correlate enzyme localization with substrate accumulation patterns .

• Developmental regulation assessment: Use immunohistochemistry with UGT89C1 antibodies across different plant developmental stages to track enzyme expression in relation to flavonoid synthesis phases .

• Stress response analysis: Employ quantitative immunoblotting to measure UGT89C1 protein levels in response to various stresses, correlating with changes in flavonoid glycosylation profiles .

• Protein complex identification: Use UGT89C1 antibodies for co-immunoprecipitation to identify protein interaction partners within the flavonoid metabolic pathway .

• Subcellular compartmentalization: Determine UGT89C1's precise subcellular localization, which is predicted to be cytosolic based on homology to related UGTs, using immunogold electron microscopy for high-resolution analysis.

These methodologies can reveal how UGT89C1 contributes to flavonoid glycosylation patterns and their biological significance in plant development and stress responses.

What strategies overcome cross-reactivity issues with UGT89C1 antibodies?

Cross-reactivity is a significant challenge when working with UGT family antibodies due to sequence conservation. To overcome this limitation, implement these advanced validation strategies:

• Competitive peptide blocking: Pre-incubate antibodies with synthetic peptides corresponding to the epitope region to confirm specificity through signal elimination .

• Knockout/knockdown controls: Validate antibody specificity using tissues from UGT89C1 mutants (e.g., ugt89c1 mutants mentioned in search results) as negative controls .

• Western blot protein panel: Test antibody against a panel of recombinant UGT proteins to assess cross-reactivity with related family members:

  UGT Family Member	Molecular Weight	Sequence Identity to UGT89C1	Expected Cross-Reactivity
  UGT89C1 (target)	51.6 kDa	100%	Strong positive
  UGT76E1	Variable	Low (different UGT group)	Minimal/None
  UGT76E2	Variable	Low (different UGT group)	Minimal/None
  UGT76E4	Variable	Low (different UGT group)	Minimal/None

• Epitope analysis: Use structural data from crystal structures of UGT89C1 to identify unique surface-exposed regions that distinguish it from other UGTs .

• Absorption validation: Perform pre-absorption of antibodies with related UGT proteins to remove cross-reactive antibodies before experimental use .

These rigorous validation steps ensure that observed signals are truly specific to UGT89C1 rather than related glycosyltransferases.

How do mutations in key residues of UGT89C1 affect antibody recognition?

Mutations in UGT89C1 can significantly impact antibody recognition depending on epitope location. This table summarizes the effects of specific mutations:

For comprehensive analysis of how mutations affect antibody binding:

• Site-directed mutagenesis: Generate a panel of UGT89C1 mutants using the Q5 Site-directed mutagenesis kit protocol as described in the research literature .

• Epitope mapping: Perform systematic epitope mapping using peptide arrays or hydrogen-deuterium exchange mass spectrometry to identify the precise binding regions of antibodies.

• Binding affinity assessment: Use surface plasmon resonance or ELISA to quantitatively compare antibody binding affinity to wild-type versus mutant proteins.

• Structural analysis: Correlate findings with the crystal structure data available for UGT89C1, particularly focusing on conformational changes induced by mutations .

This approach provides critical insights for researchers developing antibodies against specific functional domains of UGT89C1.

What are the methodological differences in using UGT89C1 antibodies for plant versus heterologous expression systems?

When using UGT89C1 antibodies across different experimental systems, researchers must adjust their methodologies:

Plant Tissue Applications:

• Tissue preparation: Requires fixation with 4% paraformaldehyde to preserve tissue architecture while maintaining antigen accessibility .

• Background reduction: Implement additional blocking steps using plant-specific blocking agents to minimize non-specific binding to endogenous plant proteins.

• Detection systems: May require signal amplification techniques (e.g., tyramide signal amplification) due to potentially lower expression levels of native UGT89C1.

• Controls: Include wild-type versus UGT89C1 mutant tissues (e.g., rol1-2 suppressor mutants of UGT89C1) as specificity controls .

Heterologous Expression Systems:

• Sample preparation: Often simpler, using direct lysis of cultured cells followed by SDS-PAGE separation.

• Tag detection: Can utilize tag-specific antibodies (e.g., anti-His) as alternative detection method for recombinant UGT89C1 .

• Expression verification: Include Western blot analysis using both tag-specific and UGT89C1-specific antibodies to confirm expression .

• Controls: Use non-transformed cells as negative controls and cells expressing related UGTs to assess specificity.

The buffer composition also differs between systems - plant tissues often require more complex extraction buffers to overcome phenolic compounds and secondary metabolites that might interfere with antibody binding.

How can UGT89C1 antibodies be used in studies of auxin transport and flavonol interactions?

UGT89C1 antibodies can provide valuable insights into the relationship between flavonol glycosylation and auxin transport through several methodological approaches:

• Co-localization with PIN proteins: Implement dual immunofluorescence labeling using UGT89C1 antibodies alongside PIN2 antibodies (1:800 dilution) with Cy3-labeled secondary antibodies (1:600 dilution) .

• Quantitative analysis of flavonol-induced PIN2 polarity changes: Use UGT89C1 antibodies to correlate enzyme levels with PIN2 localization patterns in wild-type versus UGT89C1 mutant backgrounds:

  Genotype	UGT89C1 Expression	PIN2 Polarity	Auxin Transport
  Wild-type	Normal	Predominantly basal	Normal
  rol1-2	Normal	Altered	Affected
  rol1-2 x ugt89c1	Absent	Suppressed phenotype	Rescued
  ugt89c1	Absent	Near normal	Altered auxin conjugates

• Mutant analysis workflow: Implement a systematic approach to study the functional relationship between UGT89C1 and auxin transport:

  • Generate FLS1:PID constructs for tissue-specific expression

  • Transform plants using binary vectors with appropriate selection markers

  • Perform automated whole-mount protein immunolocalization

  • Conduct polarity evaluation with blinded analysis to avoid bias

• Biochemical analysis: Use UGT89C1 antibodies in conjunction with auxin measurements to correlate enzyme levels with "levels of auxin conjugates and catabolites [that] are strongly increased in the ugt89c1 mutant background" .

This integrated approach reveals how UGT89C1-mediated flavonol rhamnosylation influences auxin transport and plant development.

What are common issues with UGT89C1 immunodetection and how can they be resolved?

When working with UGT89C1 antibodies, researchers may encounter several technical challenges:

• High background signal in plant tissues

  • Problem: Plant tissues contain phenolic compounds that can cause non-specific binding.

  • Solution: Pre-treat samples with polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) to remove phenolics before antibody incubation. Additionally, increase blocking reagent concentration (5% BSA or 10% normal serum) and include 0.1-0.3% Triton X-100 in washing buffers .

• Weak or absent UGT89C1 signal

  • Problem: Low endogenous expression levels or epitope masking.

  • Solution: Implement signal amplification using tyramide signal amplification (TSA) or increase antibody concentration. Consider antigen retrieval methods like citrate buffer treatment at 95°C for 10-15 minutes to expose masked epitopes.

• Cross-reactivity with other UGTs

  • Problem: Antibody detects related glycosyltransferases.

  • Solution: Pre-absorb antibody with recombinant proteins from related UGT family members. Use tissues from UGT89C1 knockout plants as negative controls to confirm specificity .

• Variable results between experiments

  • Problem: Inconsistent fixation or detection conditions.

  • Solution: Standardize all protocol steps and consider using automated systems like the InsituPro VSi pipetting robot for whole-mount immunolocalization .

• Inconsistent western blot results

  • Problem: Protein degradation or inefficient transfer.

  • Solution: Include protease inhibitors during extraction, optimize transfer conditions for the 51.6 kDa UGT89C1 protein, and validate with recombinant UGT89C1 as a positive control .

These troubleshooting approaches increase reliability and reproducibility of UGT89C1 antibody-based experiments.

How can UGT89C1 antibodies be validated using mutant lines and crystal structure data?

A comprehensive validation strategy for UGT89C1 antibodies combines genetic tools with structural information:

Genetic Validation Approach:

• Mutant line screening: Test antibody specificity using multiple UGT89C1 mutant lines such as:

  • rol1-2 suppressor mutants of UGT89C1 mentioned in the literature

  • T-DNA insertion lines disrupting the UGT89C1 locus

  • CRISPR/Cas9-generated knockout lines

• Expression analysis correlation: Compare immunodetection results with transcript levels determined by RT-PCR or RNA-seq to confirm correlation between protein and mRNA levels.

Structure-Based Validation:

• Epitope mapping informed by crystal structure: Use the solved crystal structure of UGT89C1 to identify surface-exposed regions uniquely characteristic of this enzyme :

  • The catalytic His21 residue

  • The donor sugar binding pocket containing Asp356, His357, Pro147, and Ile148

  • Regions distinguishing UGT89C1 from other UGTs

• Recombinant protein panel testing: Create a panel of truncated or domain-swapped recombinant proteins based on structural domains to precisely map antibody binding regions.

• Antigen competition assay: Develop synthetic peptides corresponding to key structural elements and use them in competitive binding assays to confirm epitope specificity.

• Mutational analysis: Generate specific point mutations of key residues identified in the crystal structure (His21, Asp356, His357, Pro147, Ile148) and test antibody binding to these variants .

This integrated validation strategy ensures that UGT89C1 antibodies reliably detect their intended target and can distinguish it from related enzymes.

How can UGT89C1 antibodies be adapted for high-throughput screening of glycosylation modulators?

UGT89C1 antibodies can be incorporated into high-throughput screening platforms to identify compounds that modulate glycosylation activity:

• ELISA-based activity assay: Develop a plate-based assay where:

  • Recombinant UGT89C1 is immobilized in microplate wells

  • Compounds of interest are added with UDP-β-l-rhamnose and acceptor substrates

  • Activity is measured using antibodies that detect either:
    a) Remaining UGT89C1 enzyme accessibility (conformational change)
    b) Product formation using product-specific antibodies

• Immuno-based enzymatic assay: Adapt the enzymatic assay methodology described in the research literature :

  • Use 1 mM Tris, 1 mM MgCl₂ (pH 8.0), UDP-sugars, substrates

  • Incubate at 37°C for defined timeframes

  • Detect reaction products using LC-MS combined with immunoassays

• Cellular thermal shift assay (CETSA): Apply UGT89C1 antibodies in CETSA to:

  • Assess compound binding through thermal stability shifts

  • Detect conformational changes upon ligand binding

  • Screen libraries for compounds that stabilize or destabilize UGT89C1

• Automated microscopy platform: Develop high-content imaging using:

  • Fluorescently-labeled UGT89C1 antibodies

  • Plant cells treated with compound libraries

  • Quantitative image analysis for enzyme localization or expression changes

This approach enables screening of compound libraries for modulators of flavonoid glycosylation, with potential applications in improving plant stress resistance or enhancing production of bioactive natural products.

What are the methodological considerations for using UGT89C1 antibodies in studying plant stress responses?

When studying plant stress responses using UGT89C1 antibodies, several methodological considerations are critical:

• Time-course analysis: Implement systematic sampling across stress exposure time points:

  • Early response (minutes to hours)

  • Intermediate response (hours to days)

  • Long-term adaptation (days to weeks)

• Stress-specific protocol adjustments:

  Stress Type	Sample Preparation	Antibody Concentration	Special Considerations
  Drought	Include dehydration controls	Increase by 25%	Higher background due to concentrated cellular contents
  UV stress	Protect samples from light	Standard	May need reduced fixation time
  Salt	Remove excess salts before processing	Standard	Additional washing steps
  Pathogen	Include non-infected controls	Standard	Consider pathogen-specific antibody cross-reactivity

• Comparative analysis workflow:

  • Collect stressed and control plants simultaneously

  • Process all samples in parallel using identical protocols

  • Implement blinded analysis using numbered samples to prevent bias

  • Use automated whole-mount immunolocalization for consistency

• Integration with metabolite profiling:

  • Correlate UGT89C1 protein levels with flavonol glycoside profiles

  • Compare wild-type versus UGT89C1 mutant responses

  • Consider that "levels of auxin conjugates and catabolites are strongly increased in the ugt89c1 mutant background"

• Cellular redistribution assessment:

  • Monitor potential stress-induced changes in UGT89C1 subcellular localization

  • Use co-immunoprecipitation with UGT89C1 antibodies to identify stress-specific protein interaction partners

These methodological considerations ensure reliable detection of stress-induced changes in UGT89C1 expression, localization, and activity.

What approaches combine crystallography data with antibody epitope mapping for UGT89C1?

Integrating crystallography data with antibody epitope mapping provides powerful insights into UGT89C1 structure-function relationships:

• Structure-guided epitope prediction:

  • Analyze the crystal structure of UGT89C1 to identify surface-exposed regions

  • Focus on regions with high antigenicity scores and low sequence conservation with other UGTs

  • Pay special attention to the catalytic His21 region and donor binding pocket containing Asp356, His357, Pro147, and Ile148

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with antibody binding:

  • Compare HDX-MS profiles of UGT89C1 alone versus antibody-bound UGT89C1

  • Identify protected regions that represent antibody binding sites

  • Correlate findings with structural elements identified in the crystal structure

• Site-directed mutagenesis based on crystal structure:

  • Generate point mutations of key residues identified in the crystal structure:

    • His21 (catalytic base)

    • Ser124 (non-catalytic in UGT89C1, unlike other UGTs)

    • Asp356, His357 (donor specificity)

    • Pro147, Ile148 (donor specificity)

  • Test antibody binding to these mutants to map precise epitope requirements

• Conformational epitope analysis:

  • Use molecular dynamics simulations as described in the literature to identify:

    • Flexible regions that might adopt different conformations

    • Regions that undergo conformational changes upon substrate binding

  • Test antibody binding under different conditions (with/without substrates) to identify conformation-specific antibodies

• X-ray crystallography of antigen-binding fragments (Fab):

  • Generate Fab fragments from purified antibodies

  • Co-crystallize UGT89C1 with Fab fragments

  • Determine the precise molecular interactions at the antibody-antigen interface

This integrated approach provides unprecedented insights into both antibody specificity and UGT89C1 structural dynamics.

How might UGT89C1 antibodies contribute to understanding glycosylation in diverse plant species?

UGT89C1 antibodies offer promising tools for comparative glycosylation studies across plant species:

• Cross-species epitope conservation analysis:

  • Sequence alignment of UGT89C1 orthologs across diverse plant species

  • Identification of conserved epitopes for developing broadly-reactive antibodies

  • Development of species-specific antibodies targeting variable regions

• Evolutionary glycosylation pattern studies:

  • Use UGT89C1 antibodies to track expression patterns across evolutionary distant plant species

  • Correlate enzyme distribution with glycosylation profiles and ecological adaptations

  • Compare with flavonol rhamnosylation patterns in various plant lineages

• Crop improvement applications:

  • Develop screening methods using UGT89C1 antibodies to identify varieties with enhanced glycosylation capacity

  • Use immunolocalization to map tissue-specific expression in crop plants

  • Correlate UGT89C1 expression with agriculturally valuable traits like stress resistance

• Methodology for cross-species studies:

  • Optimize fixation and antigen retrieval protocols for diverse plant tissues

  • Develop epitope-specific antibodies targeting highly conserved regions

  • Implement positive and negative controls for each species to validate antibody performance

This approach extends UGT89C1 antibody applications beyond model organisms like Arabidopsis, contributing to broader understanding of glycosylation biology across the plant kingdom.

What novel technical approaches could enhance UGT89C1 antibody specificity and sensitivity?

Emerging technologies offer opportunities to develop next-generation UGT89C1 antibodies with enhanced properties:

• Nanobody/single-domain antibody development:

  • Engineer camelid-derived nanobodies against UGT89C1

  • Advantages include smaller size for better tissue penetration and recognition of cryptic epitopes

  • Potential for direct fusion to fluorescent proteins for live-cell imaging

• Phage display selection with negative screening:

  • Implement a subtractive selection strategy:

    • Positive selection against recombinant UGT89C1

    • Negative selection against closely related UGTs

  • Further refine using structural data to target unique epitopes

• Synthetic antibody engineering:

  • Design synthetic antibodies based on structural data of UGT89C1

  • Focus on the His21 catalytic region that employs a unique catalytic dyad system

  • Target the distinct donor specificity region containing Asp356, His357, Pro147, and Ile148

• Proximity-based detection systems:

  • Develop split enzyme complementation assays where:

    • UGT89C1 antibody is fused to one enzyme fragment

    • Substrate or interaction partner is fused to complementary fragment

    • Activity occurs only when UGT89C1 is in proximity to its substrate/partner

• CRISPR-based epitope tagging:

  • Use CRISPR/Cas9 to insert small epitope tags into endogenous UGT89C1

  • Leverage well-characterized tag-specific antibodies for detection

  • Maintain native expression and regulation patterns