POFUT2 Antibody, FITC conjugated

What is POFUT2 and why is it an important research target?

POFUT2 (Protein O-Fucosyltransferase 2) is an enzyme (EC 2.4.1.221) that catalyzes the addition of O-fucose to thrombospondin type 1 repeats (TSRs) in various proteins. This post-translational modification is crucial for proper protein folding, secretion, and function of TSR-containing proteins . POFUT2 plays significant roles in developmental processes, cell signaling, and pathogen invasion mechanisms. Research on POFUT2 is particularly important in understanding glycosylation-dependent protein functions and has implications in parasitology, developmental biology, and disease research .

What are the key specifications of commercially available POFUT2 antibodies?

Available POFUT2 antibodies include polyclonal variants raised in rabbits that target specific amino acid regions of the human POFUT2 protein. Typical specifications include:

• Molecular Weight Detection: ~49-50 kDa

• Common Applications: Western blotting (WB) and immunohistochemistry (IHC)

• Species Reactivity: Primarily human and mouse, with predicted reactivity in other species (pig, zebrafish, horse, etc.)

• Available Conjugations: Including FITC (fluorescein isothiocyanate) for fluorescence applications

• UniProt ID: Q9Y2G5

What are the optimal sample preparation techniques for POFUT2 detection using FITC-conjugated antibodies?

For optimal POFUT2 detection using FITC-conjugated antibodies, researchers should consider the following protocol adaptations:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve protein structure while maintaining fluorophore activity

• Permeabilization: For intracellular targets, use 0.1-0.3% Triton X-100 for 10 minutes

• Blocking: Implement a 1-hour blocking step with 5% BSA to reduce non-specific binding

• Antibody Dilution: Determine optimal dilution empirically, typically starting at 1:50-1:200

• Incubation Conditions: Incubate overnight at 4°C in darkness to protect the FITC fluorophore

• Counterstaining: Use DAPI for nuclear visualization, avoiding propidium iodide which has spectral overlap with FITC

• Mounting: Use anti-fade mounting media specifically formulated for fluorescent preservation

Note that autofluorescence can interfere with FITC signal detection, particularly in tissues with high endogenous fluorescence. Pre-treatment with 0.1% Sudan Black B can reduce autofluorescence in such cases.

What controls should be included when working with FITC-conjugated POFUT2 antibodies?

A comprehensive control strategy for FITC-conjugated POFUT2 antibody experiments should include:

• Positive Control: Cell lines or tissues with known POFUT2 expression (e.g., peripheral blood lymphocytes for isoform A, spleen or lung tissue for isoform B)

• Negative Control: Samples lacking POFUT2 expression or POFUT2 knockout models

• Isotype Control: FITC-conjugated rabbit IgG (non-targeting) at equivalent concentration to detect non-specific binding

• Absorption Control: Pre-incubation of antibody with recombinant POFUT2 antigen to confirm specificity

• Autofluorescence Control: Unstained samples to establish baseline fluorescence

• Secondary-only Control: For comparison with directly conjugated antibody efficiency

• Signal Specificity Control: Comparison with alternative POFUT2 antibody targeting different epitopes

These controls collectively ensure signal specificity, minimize false-positive results, and provide benchmarks for quantitative analysis.

How can FITC-conjugated POFUT2 antibodies be used to investigate POFUT2 expression in different parasite development stages?

FITC-conjugated POFUT2 antibodies provide valuable tools for tracking POFUT2 expression across various parasite developmental stages, particularly in Plasmodium species. Researchers can implement the following methodological approach:

• Stage-specific Isolation: Separate parasite populations at distinct developmental stages (gametocytes, ookinetes, sporozoites)

• Immunofluorescence Analysis: Apply standard protocols with FITC-conjugated POFUT2 antibodies at 1:100 dilution

• Co-localization Studies: Combine with markers for specific organelles or structures (e.g., circumsporozoite protein)

• Quantitative Analysis: Use fluorescence intensity measurements to compare expression levels between stages

• Live Cell Imaging: For minimally invasive tracking of POFUT2 dynamics during development

This approach has revealed that POFUT2 expression varies significantly between parasite stages, with implications for understanding stage-specific glycosylation requirements. Research shows that while POFUT2 knockout parasites develop normally through asexual blood stages, they show altered phenotypes during mosquito and liver infection stages, suggesting stage-specific functional importance .

What experimental strategies can resolve contradictory findings regarding POFUT2 essentiality in Plasmodium development?

The literature presents contradictory findings regarding POFUT2 essentiality in Plasmodium development, with some studies suggesting significant fitness costs from POFUT2 deletion and others reporting minimal impact . To resolve these contradictions, researchers should consider:

• Strain Comparison Analysis: Directly compare POFUT2 knockouts in identical genetic backgrounds across multiple Plasmodium species

• Complementation Studies: Rescue experiments with wild-type and mutant POFUT2 to confirm phenotype attribution

• Conditional Knockdown Approaches: Use regulated expression systems to determine stage-specific requirements

• High-Resolution Phenotyping: Apply comprehensive phenotypic assays across all developmental stages

• Glycoproteomics Analysis: Compare O-fucosylation patterns between wild-type and POFUT2-deficient parasites

• Environmental Variation Testing: Examine outcomes under different host conditions and stress factors

Recent work suggests that differences may relate to species-specific variations between human and rodent malaria parasites, highlighting the importance of comparative approaches and careful experimental design when studying POFUT2 function across parasite species .

How can FITC-conjugated POFUT2 antibodies be integrated with other techniques to study O-fucosylation in thrombospondin repeat (TSR) domains?

FITC-conjugated POFUT2 antibodies can be integrated with complementary techniques to create a comprehensive analysis pipeline for O-fucosylation of TSR domains:

• Mass Spectrometry Integration: Combine antibody-mediated protein enrichment with glycopeptide analysis

• Click Chemistry Approaches: Use metabolic labeling with fucose analogs alongside antibody detection

• CRISPR Genetic Screening: Pair with gene editing to identify regulatory networks controlling POFUT2 activity

• Proximity Ligation Assay: Detect in situ interactions between POFUT2 and substrate proteins

• Super-Resolution Microscopy: Resolve subcellular localization of POFUT2 and modified substrates

• In Vitro Fucosylation Assays: Use recombinant POFUT2 and candidate substrates with antibody validation

This integrated approach has revealed that POFUT2-mediated O-fucosylation affects multiple TSR-containing proteins beyond the well-characterized TRAP and CSP in Plasmodium, impacting protein folding efficiency, secretion rates, and functional activity of modified proteins .

What are common challenges when using FITC-conjugated POFUT2 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with FITC-conjugated POFUT2 antibodies. These issues and their solutions include:

• Photobleaching:

  • Problem: FITC is relatively susceptible to photobleaching

  • Solution: Minimize exposure to excitation light, add anti-fade reagents to mounting media, and consider imaging FITC channels first in multi-color experiments

• Background Fluorescence:

  • Problem: High autofluorescence in the FITC channel, especially in certain tissues

  • Solution: Include Sudan Black B treatment (0.1-0.3% for 10 minutes) before antibody incubation, optimize blocking conditions, and use spectral unmixing during analysis

• Signal Specificity:

  • Problem: Uncertainty regarding signal specificity to POFUT2

  • Solution: Validate with competitive binding using recombinant POFUT2 protein (125-271AA region) and compare with alternative antibody clones

• Signal Intensity Variation:

  • Problem: Variable fluorescence intensity between experiments

  • Solution: Include calibration standards, maintain consistent exposure settings, and normalize to internal controls

• Isoform Specificity:

  • Problem: POFUT2 exists in multiple isoforms with tissue-specific expression patterns

  • Solution: Select antibodies targeting common regions or specific isoforms based on research needs and verify expression patterns in target tissues using RT-PCR

How should researchers optimize protocols for dual labeling experiments involving FITC-conjugated POFUT2 antibodies?

Dual labeling experiments involving FITC-conjugated POFUT2 antibodies require careful optimization to achieve clear signal separation and minimize crosstalk. Recommended optimization strategies include:

• Fluorophore Selection: Choose secondary fluorophores with minimal spectral overlap with FITC (e.g., Cy5, Texas Red)

• Sequential Staining: Apply FITC-conjugated POFUT2 antibody first, followed by unconjugated primary and fluorophore-conjugated secondary antibodies

• Cross-Blocking: Include species-specific blocking steps between antibody applications

• Dilution Optimization: Titrate FITC-POFUT2 antibody concentrations (typically 1:100-1:200) to minimize bleed-through

• Microscopy Settings: Utilize sequential scanning and narrow bandpass filters to minimize spectral overlap

• Controls: Include single-stained controls for spectral compensation during analysis

This approach has been successfully applied to co-localize POFUT2 with its substrates or with cellular markers to determine subcellular localization patterns across different cell types and tissues.

How might FITC-conjugated POFUT2 antibodies contribute to understanding glycosylation patterns in disease models?

FITC-conjugated POFUT2 antibodies offer unique opportunities for investigating glycosylation alterations in various disease contexts through several innovative approaches:

• Cancer Glycobiology: Tracking POFUT2 expression and localization changes in tumor progression and metastasis

• Parasite-Host Interactions: Visualizing POFUT2-dependent modifications in pathogen invasion mechanisms

• Developmental Disorders: Examining POFUT2 dysregulation in congenital glycosylation disorders

• Live-Cell Dynamics: Monitoring real-time changes in POFUT2 distribution during cellular responses

• Tissue-Specific Patterns: Mapping POFUT2 expression across healthy and diseased tissues

The fluorescent properties of FITC-conjugated antibodies enable high-resolution imaging of POFUT2 in disease models, potentially revealing therapeutic targets based on glycosylation patterns. Research in Plasmodium has already demonstrated that POFUT2-mediated modifications influence parasite invasion efficiency and may represent intervention points for novel therapeutics .

What novel experimental systems could leverage FITC-conjugated POFUT2 antibodies for functional studies?

Emerging experimental systems that could benefit from FITC-conjugated POFUT2 antibodies include:

• Microfluidic Organ-on-Chip Models: Real-time visualization of POFUT2 activity in physiologically relevant systems

• CRISPR Activation/Inhibition Screens: High-throughput imaging of POFUT2 modulation effects

• Protein Degradation Systems: Monitoring POFUT2 turnover using fluorescence decay measurements

• Glycosylation-Dependent Protein Trafficking: Tracking modified protein movement through secretory pathways

• Synthetic Biology Approaches: Engineering artificial glycosylation circuits with fluorescent readouts

• Single-Cell Glycomics: Correlating POFUT2 expression with glycan profiles at the single-cell level

• In Vivo Imaging: Using antibody-based detection systems for whole-organism studies

These approaches could elucidate fundamental questions about protein O-fucosylation dynamics and reveal new biological roles for POFUT2 beyond currently established functions.