OFUT7 Antibody

What is OFUT7 and what is its biological significance in plant research?

OFUT7 is an O-fucosyltransferase enzyme found in Arabidopsis thaliana (Mouse-ear cress) that belongs to a family of enzymes responsible for protein O-fucosylation, a post-translational modification that regulates diverse developmental processes in plants. O-fucosylation involves the addition of fucose sugar molecules to serine or threonine residues in proteins, which can significantly alter protein function and interactions .

Unlike typical ER-localized protein O-fucosyltransferases (POFUTs) found in secreted or cell surface proteins, OFUT7 belongs to a novel class of nucleocytoplasmic POFUTs that modify intracellular proteins, similar to the SPINDLY (SPY) protein in Arabidopsis thaliana . The discovery of nucleocytoplasmic O-fucosylation has dramatically expanded our understanding of this post-translational modification beyond its previously known roles in secreted proteins .

How is the OFUT7 antibody typically applied in plant biology research?

OFUT7 antibody is primarily used for detecting and studying O-fucosyltransferase expression patterns and localization in plant tissues through various techniques:

• Immunohistochemistry/Immunofluorescence: For visualizing OFUT7 distribution in plant tissues using protocols similar to those described for other plant antibodies, involving antigen retrieval in citrate buffer or Tris-EDTA at appropriate pH, followed by blocking with serum and overnight incubation with primary antibody .

• Western Blotting: For detecting OFUT7 protein in plant tissue extracts, typically using a 1:500 to 1:1000 dilution of primary antibody .

• Immunoprecipitation: For isolating OFUT7 and its interaction partners from plant cell lysates, enabling the study of protein-protein interactions and post-translational modifications .

• ELISA: For quantitative detection of OFUT7 protein levels in plant samples .

Research applications include developmental biology studies, investigation of fucosylation patterns, and analysis of mutations affecting O-fucosyltransferase activity.

What experimental controls should be included when using OFUT7 antibody?

Proper experimental controls are essential for validating OFUT7 antibody results:

These controls help distinguish true positive signals from non-specific binding or background, which is particularly important given the existence of multiple O-fucosyltransferase family members in Arabidopsis .

What are the optimal protocols for tissue preparation when using OFUT7 antibody?

When preparing plant tissues for OFUT7 antibody applications, researchers should consider these methodological approaches:

For immunohistochemistry:

• Fixation: Tissues should be placed in 10% buffered formalin for three days, followed by transfer to 70% ethanol for storage.

• Processing: Embed tissues in ParaPlast Extra for sectioning.

• Antigen retrieval: Microwave heat tissues in either pH 6 citrate buffer or pH 9 Tris-EDTA, depending on the epitope accessibility.

• Blocking: Apply 5% serum blocking buffer (e.g., horse serum) for approximately 17 minutes.

• Antibody application: Dilute OFUT7 antibody (typically 1:3 in PBS with 1.5% horse serum) and incubate overnight at 4°C .

For protein extraction and Western blotting:

• Harvest fresh plant tissues and flash-freeze in liquid nitrogen.

• Grind tissues to a fine powder while maintaining freezing conditions.

• Extract proteins using a buffer containing protease inhibitors to prevent degradation.

• Clarify extracts by centrifugation before SDS-PAGE separation.

• Transfer proteins to membranes using standard protocols, but optimize transfer time for the specific molecular weight of OFUT7 .

How can researchers optimize antibody dilution and incubation conditions?

Optimization of OFUT7 antibody protocols requires systematic testing of multiple parameters:

Recommended dilution optimization approach:

• Perform an initial titration experiment using serial dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000).

• Analyze signal-to-noise ratio at each dilution.

• Select the dilution that provides highest specific signal with minimal background.

Incubation conditions:

• Temperature: Compare results at 4°C, room temperature, and 37°C.

• Duration: Test different incubation times (1 hour, overnight, 48 hours).

• Buffer composition: Optimize blocking agents (BSA, normal serum, casein) and detergent concentration.

For quantitative applications like ELISA, generate a standard curve using purified recombinant OFUT7 protein to determine the linear range of antibody response and establish detection limits .

What are the challenges in detecting OFUT7 in different plant tissues?

Detecting OFUT7 across various plant tissues presents several methodological challenges:

• Variable expression levels: OFUT7 may be expressed at different levels across tissues and developmental stages, requiring adaptation of detection protocols.

• Fixation artifacts: Overfixation can mask epitopes, while underfixation risks tissue degradation. For consistent results, standardize fixation times and conditions.

• Tissue-specific autofluorescence: Plant tissues contain compounds that exhibit natural fluorescence, potentially interfering with immunofluorescence detection. Control for this by imaging unstained samples to identify autofluorescence patterns.

• Extraction efficiency: Different plant tissues require specific protein extraction protocols due to varying compositions of cell wall components, secondary metabolites, and proteases.

• Cross-reactivity with other O-fucosyltransferases: The OFUT family in Arabidopsis includes multiple members (OFUT4, OFUT7, OFUT13, OFUT16, etc.), requiring verification of antibody specificity in each tissue type .

To address these challenges, researchers should perform tissue-specific optimizations and include appropriate controls for each experimental context.

How does the structure of OFUT7 relate to its enzymatic function?

Understanding the structure-function relationship of OFUT7 provides insights into its enzymatic mechanism:

Structural studies using cryo-electron microscopy (cryo-EM) of related O-fucosyltransferases reveal that:

• The enzyme forms an antiparallel dimer rather than the X-shaped dimer observed in human OGT.

• The catalytic domain can interconvert among multiple conformations.

• The N-terminal disordered peptide in these enzymes contains trans auto-fucosylation sites and may inhibit POFUT activity.

• TPRs 1-5 dynamically regulate enzymatic activity by interfering with protein substrate binding .

These structural insights can guide experimental design when using OFUT7 antibody to investigate the enzyme's localization and interaction partners, particularly when studying mutations that might affect its structure and function.

What approaches can be used to study OFUT7's role in plant developmental processes?

To investigate OFUT7's role in plant development, researchers can employ multiple complementary approaches:

• Genetic manipulation:

  • CRISPR/Cas9 gene editing to create OFUT7 knockout or site-specific mutations

  • RNAi for tissue-specific or inducible knockdown

  • Overexpression studies using constitutive or tissue-specific promoters

• Protein-protein interaction studies:

  • Co-immunoprecipitation with OFUT7 antibody to identify interaction partners

  • Yeast two-hybrid screening

  • Proximity labeling methods (BioID, APEX)

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction validation

• Fucosylation pattern analysis:

  • Mass spectrometry to identify O-fucosylated proteins

  • Site-directed mutagenesis of putative fucosylation sites

  • Generation of fucosylation-specific antibodies

• Developmental phenotyping:

  • Time-course immunohistochemistry using OFUT7 antibody across developmental stages

  • Correlation of OFUT7 expression with specific developmental markers

  • Phenotypic analysis of OFUT7 mutants during plant development

These approaches can be combined to create a comprehensive understanding of OFUT7's developmental functions.

How can researchers distinguish between OFUT7 and other O-fucosyltransferase family members?

Distinguishing between closely related O-fucosyltransferase family members requires multiple strategies:

• Epitope mapping: Determine the exact epitope recognized by the OFUT7 antibody through:

  • Peptide arrays covering unique regions of OFUT7

  • Competition assays with synthetic peptides

  • Alanine scanning mutagenesis of potential epitopes

• Cross-reactivity testing:

  • Express recombinant proteins for each OFUT family member

  • Perform Western blot analysis with the OFUT7 antibody

  • Create a cross-reactivity profile table documenting binding to each family member

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Perform IP with OFUT7 antibody

  • Analyze precipitated proteins by MS to confirm specificity

  • Identify any co-precipitated proteins from the OFUT family

• Genetic verification:

  • Use tissues from knockout lines of various OFUT family members

  • Confirm presence/absence of antibody signal in each genetic background

This multi-faceted approach ensures that experimental observations are correctly attributed to OFUT7 rather than other family members.

What are common causes of false positives/negatives when using OFUT7 antibody?

When using OFUT7 antibody, researchers should be aware of several potential sources of experimental artifacts:

Common causes of false positives:

• Cross-reactivity with other O-fucosyltransferase family members (OFUT4, OFUT13, etc.)

• Endogenous peroxidase activity in plant tissues if using HRP-conjugated detection systems

• Non-specific binding to highly abundant proteins

• Sticky protein aggregates formed during sample preparation

• Matrix effects when using plant extracts in immunoassays

Common causes of false negatives:

• Epitope masking due to protein folding or interaction with other proteins

• Degradation of OFUT7 protein during sample preparation

• Insufficient antigen retrieval in fixed tissues

• Post-translational modifications altering antibody recognition

• Low expression levels below detection threshold

Mitigation strategies:

• Use multiple antibody dilutions and exposure times

• Include genetic controls (OFUT7 knockout/overexpression)

• Verify results with alternative detection methods

• Perform reciprocal experiments (gain-of-function and loss-of-function)

How should researchers interpret contradictory data when studying OFUT7 expression?

When confronted with contradictory data regarding OFUT7 expression or function, consider these analytical approaches:

The complexity of plant development and cellular contexts often explains apparent contradictions in experimental results.

What are the best approaches for quantifying OFUT7 expression levels?

Accurate quantification of OFUT7 expression requires optimization of several methodological approaches:

Western blot quantification:

• Use internal loading controls (housekeeping proteins)

• Apply densitometry analysis with software like ImageJ

• Create standard curves using recombinant OFUT7 protein

• Ensure linear detection range through serial dilutions

ELISA-based quantification:

• Develop a sandwich ELISA using two antibodies recognizing different OFUT7 epitopes

• Generate a standard curve with purified recombinant OFUT7

• Optimize blocking conditions to reduce background

• Validate with samples of known concentration

Quantitative immunohistochemistry:

• Use consistent image acquisition parameters

• Apply digital image analysis to measure staining intensity

• Include calibration controls in each experiment

• Normalize to cell number or tissue area

Comparison of protein vs. mRNA quantification:

• Correlate antibody-based protein measurements with qRT-PCR data

• Consider post-transcriptional regulation if discrepancies exist

• Evaluate protein stability through cycloheximide chase experiments

How can active learning approaches improve OFUT7 antibody-based research?

Active learning strategies can significantly enhance OFUT7 antibody-based research by optimizing experimental design and data interpretation:

Active learning refers to computational approaches that iteratively select the most informative experiments to perform next, based on existing data. For antibody-antigen interactions, specific active learning strategies have been shown to reduce the number of required experiments by up to 35% .

Implementation approach for OFUT7 research:

• Start with a limited dataset of OFUT7 antibody binding experiments

• Apply machine learning models to predict binding patterns

• Identify the most informative next experiments to validate predictions

• Iteratively refine predictions with new experimental data

Practical applications:

• Optimization of antibody dilutions and conditions

• Screening of tissue samples for OFUT7 expression

• Epitope mapping to improve antibody specificity

• Identification of cross-reactivity with other O-fucosyltransferases

This approach is particularly valuable when working with limited research materials or when screening large numbers of experimental conditions .

What are the latest methods for studying O-fucosylation patterns mediated by OFUT7?

Recent methodological advances have enhanced our ability to study O-fucosylation patterns:

• Mass spectrometry approaches:

  • Electron transfer dissociation (ETD) MS for precise site identification

  • Quantitative glycoproteomics using isobaric labeling

  • Targeted parallel reaction monitoring (PRM) for specific O-fucosylated peptides

  • Native MS to preserve intact glycoprotein structures

• Fucose-specific labeling:

  • Metabolic labeling with fucose analogs containing bioorthogonal handles

  • Click chemistry-based visualization of fucosylated proteins

  • Proximity labeling of proteins near active O-fucosyltransferases

• Advanced microscopy:

  • Super-resolution imaging of fucosylated proteins using specific lectins

  • Correlative light and electron microscopy to precisely localize O-fucosylation

  • FRET-based sensors to monitor O-fucosylation in living cells

• Computational approaches:

  • Prediction algorithms for O-fucosylation sites

  • Molecular dynamics simulations of OFUT7-substrate interactions

  • Systems biology models integrating O-fucosylation with other post-translational modifications

These methods can be combined to create a comprehensive picture of OFUT7-mediated O-fucosylation in plant development.

How can researchers develop anti-idiotypic antibodies for studying OFUT7?

Anti-idiotypic antibodies can serve as valuable tools for studying OFUT7 function and interactions:

Anti-idiotypic antibodies bind to the idiotope (unique antigen binding site) of another antibody. For OFUT7 research, these specialized antibodies can be developed to:

• Mimic structural features of OFUT7 substrates

• Block or modulate OFUT7 antibody binding

• Serve as surrogate antigens in assay development

Development methodology:

• Selection strategy: Generate anti-idiotypic antibodies by performing selection on the original OFUT7 antibody in the presence of isotype-matched antibodies as blockers to ensure idiotope specificity .

• Types of anti-idiotypic antibodies to develop:

  • Type 1 (inhibitory): These bind at or near the antigen-binding site of the OFUT7 antibody and can be used in cell-based assays and ELISA.

  • Type 2 (non-inhibitory): These bind to an idiotope outside the antigen-binding site and can detect both free and bound OFUT7 antibody.

  • Type 3 (complex-specific): These recognize the OFUT7 antibody-antigen complex specifically .

• Applications in OFUT7 research:

  • Pharmacokinetic studies of OFUT7 antibody in plant systems

  • Development of surrogate assays when native OFUT7 protein is limited

  • Investigation of OFUT7 binding mechanisms

  • Creation of standardized positive controls for assay validation

This approach expands the toolkit available for studying OFUT7 expression and function in complex plant systems.