OFUT19 Antibody

What is OFUT19 Antibody and what is its target in research applications?

OFUT19 antibody is a polyclonal antibody developed against Arabidopsis thaliana O-fucosyltransferase 19 (OFUT19), an enzyme involved in protein fucosylation. The target protein (OFUT19, UniProt ID: Q9SH89) is classified as an O-fucosyltransferase family protein that catalyzes the transfer of fucose residues to proteins . This antibody is primarily used in plant biology research for:

• Detection of OFUT19 protein expression

• Localization studies in plant tissues

• Analysis of protein-protein interactions involving OFUT19

• Investigation of O-fucosylation pathways in plant development

The antibody specifically recognizes epitopes on the OFUT19 protein and is typically produced in rabbit hosts, making it suitable for various immunological applications in research settings .

How should researchers design experiments to validate OFUT19 Antibody specificity?

Proper validation of OFUT19 antibody specificity is critical for reliable research outcomes. Researchers should implement the following comprehensive validation strategy:

Recommended Validation Protocol:

• Western Blotting Validation:

  • Test against wild-type tissues (positive control)

  • Test against OFUT19 knockout/mutant tissues (negative control)

  • Confirm single band at expected molecular weight (~EC 2.4.1 family range)

• Pre-absorption Tests:

  • Pre-incubate antibody with purified recombinant OFUT19 protein

  • Compare staining patterns with and without pre-absorption

  • Signal should be significantly reduced in pre-absorbed samples

• Cross-reactivity Assessment:

  • Test against closely related O-fucosyltransferase family members (OFUT13, OFUT16)

  • Establish specificity limits across species if conducting comparative studies

"Always use flow validated antibodies whenever possible. But where to find such information, and why would be it be so important before the experiment starts?" . This principle applies to all antibody work, including with OFUT19 antibody. Validation data should be documented comprehensively before proceeding with experimental applications.

What controls are essential when using OFUT19 Antibody in immunological applications?

Appropriate controls are fundamental for accurate interpretation of results when using OFUT19 antibody. The following controls should be implemented based on established immunological principles:

"Use an appropriate blocker to mask non-specific binding sites and lower backgrounds to improve the all-important signal-to-noise ratio" . For plant tissues, 3-5% BSA with 0.1% normal serum from the same host species as the secondary antibody (but not rabbit) is recommended for reducing background staining.

What are the optimal methodological approaches for using OFUT19 Antibody in different applications?

OFUT19 antibody can be utilized across multiple experimental platforms, each requiring specific methodological considerations:

Western Blotting Protocol:

• Protein extraction using plant-specific buffers containing protease inhibitors

• 10-12% SDS-PAGE separation

• Transfer to nitrocellulose or PVDF membrane (0.45μm)

• Blocking with 5% non-fat milk in TBST

• Primary antibody dilution: 1:1000-1:2000 (optimize empirically)

• Incubation: Overnight at 4°C

• Detection: HRP-conjugated anti-rabbit secondary antibody (1:5000)

Immunohistochemistry Protocol:

• Fixation: 4% paraformaldehyde in PBS (pH 7.4)

• Antigen retrieval: Citrate buffer (pH 6.0), microwave treatment

• Blocking: 3% BSA, 5% normal goat serum in PBS

• Primary antibody dilution: 1:100-1:500

• Incubation: Overnight at 4°C

• Detection: Fluorescently-labeled secondary antibody or DAB development

"Know your primary antibody – clonality (monoclonal/polyclonal), what is the host species (important if using secondary antibodies for increased signals), target specificity, purity, cross-reactivity with closely related proteins, and epitope recognition site" .

How does sample preparation affect OFUT19 Antibody performance in plant tissue studies?

Sample preparation significantly impacts OFUT19 antibody performance and requires careful attention to preserve both antigen integrity and accessibility:

Critical Factors in Sample Preparation:

• Fixation Method:

  • Over-fixation can mask epitopes

  • Under-fixation can compromise tissue morphology

  • Recommended: 4% paraformaldehyde, 4-16 hours at 4°C

• Permeabilization:

  • Plant cell walls require additional permeabilization

  • Options include: 0.1-0.5% Triton X-100, enzymatic digestion with cellulase/pectinase

  • Duration must be optimized for tissue type

• Storage Conditions:

  • Fresh tissues yield optimal results

  • For longer-term storage, flash freeze in liquid nitrogen

  • For sectioned tissues, store at -20°C with desiccant

  • "Cells frozen down in PBS can be stored at -20°C for at least one week before analysis"

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Enzymatic retrieval using proteinase K (1-10 μg/mL)

  • Method selection depends on fixation duration and tissue type

Sample viability assessment before immunostaining is crucial: "Perform a cell count and viability check before starting with your sample preparation. Dead cells give a high background scatter and may show false positive staining. Ensure that the cell viability is >90%" .

What advanced analytical techniques can enhance OFUT19 protein localization studies?

Researchers investigating subcellular localization of OFUT19 can employ several advanced techniques:

Super-resolution Microscopy Approaches:

• Structured Illumination Microscopy (SIM): Achieves 100-120nm resolution

• Stimulated Emission Depletion (STED): Reaches 20-50nm resolution

• Single Molecule Localization Microscopy (PALM/STORM): Provides 10-20nm precision

Co-localization Analysis Protocol:

• Double immunolabeling with OFUT19 antibody and organelle markers

• Image acquisition with identical parameters

• Quantitative co-localization analysis using:

  • Pearson's correlation coefficient

  • Mander's overlap coefficient

  • Object-based co-localization methods

CLEM (Correlative Light and Electron Microscopy):

• Immunofluorescence with OFUT19 antibody

• Sample processing for EM with gold-conjugated secondary antibodies

• Correlation of fluorescence and EM images for precise localization

"It's essential to have the right tools to understand the biology" . These advanced techniques provide researchers with enhanced spatial resolution to precisely determine OFUT19 localization within plant cellular compartments.

How can researchers troubleshoot weak or non-specific signals when using OFUT19 Antibody?

When encountering issues with OFUT19 antibody performance, systematic troubleshooting is essential:

Common Problems and Solutions:

"Antibodies successfully tested on applications such as Western Blotting or Immunohistochemistry may not be suitable for Flow cytometry analysis!" . This principle applies across techniques - validation in one application doesn't guarantee performance in another.

How should researchers quantitatively assess OFUT19 expression levels?

Accurate quantification of OFUT19 expression requires rigorous methodology:

Western Blot Quantification Protocol:

• Include concentration gradient of recombinant OFUT19 protein standard

• Process experimental samples alongside standards

• Use housekeeping proteins (actin, tubulin, GAPDH) as loading controls

• Analyze band intensity using densitometry software

• Generate standard curve and calculate relative expression

qPCR Correlation Analysis:

• Extract RNA from same samples used for protein analysis

• Perform RT-qPCR with OFUT19-specific primers

• Calculate correlation between mRNA and protein levels

• Assess potential post-transcriptional regulation

Developmental Expression Profile:

When assessing expression across developmental stages, use:

• Consistent protein extraction methodology

• Equal protein loading confirmed by Bradford assay

• Multiple biological and technical replicates

• Statistical analysis of quantification data

"To understand a test's accuracy, scientists look at sensitivity and specificity" . These principles apply to OFUT19 quantification as well - researchers should establish the detection limits and dynamic range of their quantification method.

What considerations should researchers make when using OFUT19 Antibody in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with OFUT19 antibody requires careful optimization:

Optimized Co-IP Protocol:

• Lysis Buffer Selection:

  • Use non-denaturing buffer to preserve protein-protein interactions

  • Include protease/phosphatase inhibitors

  • Consider mild detergents (0.5% NP-40 or 1% Triton X-100)

• Pre-clearing Step:

  • Incubate lysate with protein A/G beads

  • Remove non-specific binding proteins

  • Reduce background in final results

• Antibody Coupling:

  • Direct coupling to beads prevents heavy chain interference

  • Use crosslinking reagents (BS3 or DSS)

  • Alternatively, use antibody isotype not recognized by secondary antibody

• Controls:

  • IgG control from same species

  • Input sample (5-10% of starting material)

  • Reverse Co-IP if antibody for interacting protein is available

"The technology used to isolate the antibody, termed Ig-Seq, gives researchers a closer look at the antibody response" . Similarly, optimized Co-IP methods provide clearer insights into OFUT19 protein interactions.

How can researchers interpret contradictory results from different lots of OFUT19 Antibody?

Antibody lot-to-lot variation is a significant challenge requiring systematic investigation:

Investigation Strategy:

• Document Differences:

  • Record exact experimental conditions

  • Note lot numbers and handling differences

  • Quantify the degree of variation observed

• Side-by-Side Comparison:

  • Run parallel experiments with both lots

  • Include consistent positive and negative controls

  • Document all variables kept constant

• Epitope Analysis:

  • Determine if different lots recognize distinct epitopes

  • Perform epitope mapping if resources allow

  • Consider if protein modifications might affect recognition

• Validation Approaches:

  • Test with recombinant OFUT19 protein

  • Verify with genetic approaches (knockdown/knockout)

  • Consider alternative antibodies from different vendors

"We thought this was an urgent medical need, and the usual supply chains were unreliable, so we decided to build our own" . While this quote refers to COVID-19 testing, the principle applies to antibody research - when commercial reagents show inconsistency, researchers may need to develop in-house validation systems or even generate their own antibodies.

What are the latest methodological advances in antibody-based detection systems applicable to OFUT19 research?

Recent technological innovations offer enhanced detection capabilities for OFUT19 research:

Advanced Detection Technologies:

• Proximity Ligation Assay (PLA):

  • Detects protein interactions with single-molecule sensitivity

  • Visualizes OFUT19 interactions with potential substrates

  • Generates fluorescent signals only when proteins are <40nm apart

• Single-Cell Proteomics:

  • Mass cytometry (CyTOF) for single-cell protein quantification

  • Imaging mass cytometry for spatial proteomic analysis

  • DNA-barcoded antibodies for ultra-high-throughput analysis

• Microfluidic Antibody Capture:

  • Microfluidic devices for automated immunoassays

  • Reduced sample volume requirements

  • Higher throughput and reproducibility

• Machine Learning Applications:

  • "Active learning can reduce costs by starting with a small labeled subset of data and iteratively expanding the labeled dataset"

  • Algorithms improve specificity of antibody-based detection

  • Automated image analysis enhances quantification precision

These technological advances can be applied to OFUT19 research to increase sensitivity, specificity, and throughput of detection methods.

How can researchers develop experiments to study OFUT19's role in fucosylation pathways?

Investigating OFUT19's enzymatic function requires multifaceted experimental approaches:

Functional Characterization Strategy:

• In Vitro Enzymatic Assays:

  • Express and purify recombinant OFUT19

  • Develop fucosyltransferase activity assay with labeled donor substrates

  • Identify potential protein substrates through candidate approaches

• Substrate Identification:

  • Immunoprecipitate OFUT19 and identify interacting proteins by mass spectrometry

  • Perform glycoproteomic analysis to identify fucosylated proteins

  • Compare wild-type and OFUT19 mutant glycoproteomes

• Genetic Approaches:

  • Generate CRISPR/Cas9 knockout or RNAi knockdown lines

  • Perform phenotypic analysis across developmental stages

  • Complement with wild-type or catalytically inactive OFUT19

• Localization Studies:

  • Co-localize OFUT19 with Golgi/ER markers

  • Track movement of fluorescently tagged OFUT19

  • Correlate localization with protein fucosylation patterns

"This finding tells us that by focusing on antibodies that target these highly conserved sites on the spike protein, there is a way to overcome the virus' continual evolution" . Similarly, focusing on conserved functional domains of OFUT19 may reveal essential roles in plant biology.