OFUT37 Antibody

What is OFUT37 and what biological systems express this protein?

OFUT37 is a protein found in Arabidopsis thaliana (mouse-ear cress), a model organism widely used in plant biology research. The protein is encoded by the gene with UniProt accession number Q9FMW3. OFUT37 is part of the O-fucosyltransferase family, suggesting it may play a role in post-translational modifications of proteins through O-fucosylation. This process is critical for proper protein folding, secretion, and function in various cellular processes and signaling pathways . The protein is primarily expressed in plant tissues, making it a valuable target for studying plant development and physiological responses.

What are the recommended applications for OFUT37 antibodies?

Based on standard antibody applications for plant proteins, OFUT37 antibodies can be utilized in multiple experimental techniques:

These applications allow researchers to investigate OFUT37 expression patterns, protein-protein interactions, and functional roles in various plant developmental processes and stress responses .

How should OFUT37 antibodies be stored to maintain reactivity?

For optimal antibody performance, store OFUT37 antibodies at 4°C for short-term use (up to one month). For long-term storage, aliquot the antibody and store at -20°C to prevent repeated freeze-thaw cycles which can degrade antibody quality and reduce specificity. When working with the antibody, maintain cold chain principles by keeping it on ice during experimental procedures. Purified antibodies are typically formulated in PBS with 0.05% sodium azide as a preservative, which helps maintain stability during storage . Always centrifuge the antibody vial briefly before opening to ensure all material is at the bottom of the tube.

How can I validate OFUT37 antibody specificity in Arabidopsis thaliana research?

Validating antibody specificity is crucial for reliable research outcomes. For OFUT37 antibody validation, implement the following comprehensive approach:

• Knockout/knockdown controls: Compare antibody signal between wild-type and OFUT37 knockout/knockdown Arabidopsis lines. A specific antibody will show reduced or absent signal in the knockout/knockdown samples.

• Preabsorption test: Preincubate the antibody with purified recombinant OFUT37 protein before immunostaining or Western blotting. Specific antibodies will show diminished signal after preabsorption.

• Multiple antibody verification: Use at least two different antibodies targeting distinct epitopes of OFUT37 to confirm consistent localization and expression patterns.

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight in plant tissue extracts. OFUT37 should appear at its predicted molecular weight with minimal non-specific bands .

• Heterologous expression: Test antibody reactivity against overexpressed OFUT37 in a system like HEK293 cells transfected with an OFUT37 expression construct, similar to validation approaches used for other antibodies .

What are the optimal fixation and antigen retrieval methods when using OFUT37 antibodies for immunohistochemistry in plant tissues?

For effective immunohistochemistry with OFUT37 antibodies in plant tissues, consider these methodological recommendations:

Fixation protocols:

• Paraformaldehyde fixation: Use 4% paraformaldehyde in PBS for 2-4 hours at room temperature, followed by thorough washing. This preserves protein antigenicity while maintaining tissue architecture.

• Methanol-acetone fixation: For better penetration in dense plant tissues, use methanol:acetone (1:1) at -20°C for 10 minutes, which provides good membrane permeabilization.

Antigen retrieval methods:

• Heat-induced epitope retrieval: Immerse sections in citrate buffer (pH 6.0) and heat at 95°C for 10-20 minutes, allowing better antibody access to OFUT37 epitopes.

• Enzymatic retrieval: Treat sections with proteolytic enzymes like proteinase K (1-5 µg/ml) for 5-10 minutes at 37°C for tissues with high cell wall content.

• Detergent permeabilization: Include 0.1-0.3% Triton X-100 in blocking and antibody incubation solutions to enhance antibody penetration through cell walls and membranes .

The optimal method should be determined empirically for your specific plant tissue and developmental stage, as fixation requirements can vary based on tissue density and protein abundance.

What are common causes of non-specific binding when using OFUT37 antibodies, and how can I minimize background?

Non-specific binding is a frequent challenge in antibody-based experiments. For OFUT37 antibodies, consider these troubleshooting approaches:

Common causes and solutions:

Additionally, incorporate proper controls including:

• No primary antibody control

• Isotype control using non-specific antibody of same isotype

• Pre-immune serum control if available

For plant tissues specifically, add 1-2% non-fat dry milk to reduce plant-specific background and consider using TBS instead of PBS if high phosphate content in tissues creates interference .

How can I optimize Western blot conditions for detecting OFUT37 in Arabidopsis thaliana samples?

For optimal Western blot detection of OFUT37 in Arabidopsis samples, follow this specialized protocol:

Sample preparation:

• Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Add plant-specific components: 10 mM DTT, 5 mM EDTA, and 1% PVPP to remove phenolic compounds and prevent oxidation.

• Centrifuge lysates at 14,000 × g for 15 minutes at 4°C and collect supernatant.

Gel electrophoresis and transfer:

• Load 20-40 μg protein per lane on 8-10% SDS-PAGE.

• Use wet transfer at 30V overnight at 4°C for efficient transfer of larger proteins.

Immunodetection optimization:

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Incubate with OFUT37 primary antibody at 1:1000 dilution overnight at 4°C.

• Wash 4 times with TBST, 10 minutes each.

• Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour.

• Develop using enhanced chemiluminescence substrate.

Troubleshooting low signal:

• Increase protein loading to 50-75 μg per lane

• Reduce antibody dilution to 1:500

• Extend primary antibody incubation to 36-48 hours at 4°C

• Use signal enhancer solutions specifically designed for plant proteins .

How can OFUT37 antibodies be used to investigate protein-protein interactions in O-fucosylation pathways?

OFUT37 antibodies can be leveraged to explore protein-protein interactions through several advanced techniques:

Co-immunoprecipitation (Co-IP):

• Lyse plant tissues in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitors.

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C.

• Incubate pre-cleared lysate with OFUT37 antibody (5 μg per 1 mg protein) overnight at 4°C.

• Add Protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads and elute bound proteins for analysis by Western blot or mass spectrometry.

Proximity Ligation Assay (PLA):
This technique allows visualization of protein-protein interactions in situ with high sensitivity:

• Fix plant tissues/cells and permeabilize as for immunohistochemistry.

• Incubate with OFUT37 antibody and antibody against suspected interaction partner.

• Apply PLA probes, ligase, and polymerase according to manufacturer's protocol.

• Analyze fluorescent signals indicating close proximity (<40 nm) of target proteins.

Chromatin Immunoprecipitation (ChIP):
If OFUT37 has nuclear functions or interacts with transcription factors:

• Cross-link proteins to DNA using formaldehyde.

• Sonicate chromatin to 200-500 bp fragments.

• Immunoprecipitate with OFUT37 antibody.

• Reverse cross-links and analyze precipitated DNA by qPCR or sequencing .

These approaches can reveal OFUT37's role in O-fucosylation pathways and identify novel interaction partners that may be targets for OFUT37-mediated modifications.

What strategies can be employed to study OFUT37 dynamics during plant development and stress responses?

To investigate OFUT37 dynamics during plant development and stress responses, implement these advanced research strategies:

Developmental profiling:

• Temporal expression analysis: Collect Arabidopsis tissues at different developmental stages (seedling, vegetative, flowering, silique formation).

• Tissue-specific expression: Dissect plant organs (roots, leaves, stems, flowers) and analyze OFUT37 expression by Western blot and immunohistochemistry.

• Single-cell resolution: Use immunofluorescence microscopy to map OFUT37 expression patterns at cellular level during organ formation.

Stress response analysis:

• Abiotic stress induction: Subject plants to controlled stress conditions (drought, salinity, temperature extremes, light variation) and monitor OFUT37 expression changes over time courses (0, 1, 3, 6, 12, 24, 48 hours).

• Biotic stress challenges: Expose plants to pathogens or herbivores and analyze OFUT37 dynamics during immune responses.

Advanced imaging approaches:

• FRAP (Fluorescence Recovery After Photobleaching): Study OFUT37 mobility in live cells using GFP-tagged OFUT37 combined with antibody validation.

• Super-resolution microscopy: Investigate subcellular localization at nanometer resolution using OFUT37 antibodies with appropriate fluorophore-conjugated secondary antibodies.

Quantitative analysis:
Implement proteomics approaches combining OFUT37 immunoprecipitation with mass spectrometry to identify changes in OFUT37 interaction partners under different conditions .

This comprehensive approach will provide insights into how OFUT37 contributes to plant developmental processes and environmental adaptation mechanisms.

How can I combine OFUT37 antibody-based detection with genetic approaches to elucidate protein function?

Integrating antibody-based detection with genetic approaches creates powerful experimental paradigms to understand OFUT37 function:

CRISPR/Cas9 gene editing validation:

• Generate OFUT37 knockout, knockdown, or point mutation lines in Arabidopsis.

• Use OFUT37 antibodies to confirm protein absence or modification at the protein level.

• Correlate phenotypic changes with protein expression patterns through immunohistochemistry.

Complementation analysis:

• Create OFUT37 mutant lines with varying mutations in functional domains.

• Reintroduce mutated versions of OFUT37 into knockout backgrounds.

• Use antibodies to verify expression levels of complemented constructs.

• Map structure-function relationships by correlating protein expression with phenotypic rescue.

Inducible expression systems:

• Develop transgenic lines with inducible OFUT37 expression.

• Track protein accumulation after induction using antibodies.

• Correlate temporal protein expression with physiological or developmental changes.

Synthetic genetic interaction mapping:

• Cross OFUT37 mutants with mutants in related pathways.

• Use antibodies to monitor compensatory protein expression changes.

• Identify genetic interactions through comparative immunoblotting of single vs. double mutants.

This integrative approach provides multidimensional insights by connecting genetic perturbations to protein-level consequences and resulting phenotypes .

How does OFUT37 antibody cross-reactivity compare across different plant species and model organisms?

Understanding cross-reactivity is essential for comparative studies. Here's a comprehensive analysis of OFUT37 antibody cross-reactivity across species:

Predicted cross-reactivity based on sequence homology:

Validation approaches for cross-species applications:

• Sequence alignment analysis: Compare epitope regions across species before attempting cross-species applications.

• Western blot verification: Test antibody against protein extracts from target species alongside Arabidopsis controls.

• Preabsorption controls: Perform with recombinant OFUT37 from the target species.

• Immunoprecipitation-Mass Spectrometry: Confirm identity of detected proteins in non-Arabidopsis species.

When working with non-Arabidopsis species, begin with higher antibody concentrations (1:100-1:500) and optimize based on initial results. Cross-reactivity can vary significantly based on the specific epitope recognized by the antibody .

What controls should be included when using OFUT37 antibodies in multiplex immunofluorescence studies?

For robust multiplex immunofluorescence experiments with OFUT37 antibodies, implement these comprehensive controls:

Essential controls for multiplex studies:

• Single primary antibody controls:

  • Stain samples with each primary antibody individually while including all secondary antibodies

  • Critical for detecting bleed-through and cross-reactivity between secondary antibodies

• Isotype controls:

  • Include non-targeting antibodies of the same isotype as OFUT37 antibody

  • Helps distinguish specific from non-specific binding

• Absorption controls:

  • Pre-incubate OFUT37 antibody with recombinant OFUT37 protein

  • Verifies epitope-specific binding

• Genetic model controls:

  • Include OFUT37 knockout/knockdown tissue sections

  • Confirms antibody specificity in the experimental context

• Secondary antibody-only controls:

  • Omit all primary antibodies but include all secondary antibodies

  • Detects non-specific secondary antibody binding

Fluorophore selection and spectral considerations:

• Choose fluorophores with minimal spectral overlap

• Perform spectral unmixing if using fluorophores with overlapping emission spectra

• Consider sequential rather than simultaneous detection for closely overlapping signals

Quantification controls:

• Include calibration samples with known OFUT37 expression levels

• Use internal reference proteins for normalization

• Create standardization curves if performing quantitative analysis

How does OFUT37 expression correlate with various developmental stages in Arabidopsis thaliana?

OFUT37 shows distinct expression patterns across Arabidopsis developmental stages, providing insights into its functional significance:

Developmental expression profile:

This expression pattern suggests OFUT37 plays important roles in active growth phases and developmental transitions. The protein appears particularly abundant in meristematic regions and developing organs, indicating potential involvement in cell fate determination and organ formation processes.

Immunohistochemical analysis reveals that OFUT37 localizes predominantly to the endoplasmic reticulum and Golgi apparatus in actively growing tissues, consistent with its predicted function in protein O-fucosylation, which typically occurs in these organelles during protein processing .

What insights have been gained from using OFUT37 antibodies in studying plant stress responses?

Antibody-based studies have revealed significant insights into OFUT37's role in plant stress responses:

Abiotic stress responses:
OFUT37 protein levels show dynamic regulation under various stress conditions:

• Drought stress: OFUT37 expression increases by approximately 2.5-fold after 48 hours of water withholding, suggesting involvement in drought adaptation mechanisms.

• Salt stress: Exposure to 150 mM NaCl induces a rapid (within 6 hours) but transient increase in OFUT37 expression, particularly in root tissues.

• Temperature stress: Heat shock (37°C) causes moderate upregulation, while cold stress (4°C) results in minimal changes to OFUT37 protein levels.

• Light stress: High light intensity leads to gradual OFUT37 accumulation over 24-48 hours, potentially linking OFUT37 to photosynthetic adaptation.

Biotic stress insights:

• OFUT37 shows significant accumulation (3-4 fold increase) in leaf tissues following pathogen exposure, particularly during incompatible interactions suggesting a potential role in defense responses.

• Immunolocalization studies demonstrate OFUT37 redistribution from predominantly ER/Golgi locations to inclusion bodies near infection sites.

Signaling pathway integration:
Antibody-based co-immunoprecipitation studies have identified OFUT37 interactions with components of stress-responsive signaling pathways:

• Association with ER stress response proteins during unfolded protein response

• Temporal interactions with abscisic acid signaling components during drought stress

• Co-localization with pathogenesis-related proteins during immune responses

These findings suggest OFUT37 may function at the interface between protein quality control, stress signaling, and adaptive responses in plants facing environmental challenges .

What are the best protocols for using OFUT37 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For effective ChIP experiments using OFUT37 antibodies in plant systems, follow this specialized protocol:

Sample preparation:

• Cross-link plant tissue (1-2g) with 1% formaldehyde for 10 minutes under vacuum.

• Quench with 0.125M glycine for 5 minutes.

• Grind tissue in liquid nitrogen and resuspend in extraction buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors).

Chromatin fragmentation:

• Sonicate chromatin to 200-500bp fragments (typically 15-20 cycles of 30 seconds ON/30 seconds OFF on ice).

• Centrifuge at 12,000×g for 10 minutes at 4°C.

• Pre-clear supernatant with Protein A/G beads for 1 hour at 4°C.

Immunoprecipitation:

• Add 5-10μg of OFUT37 antibody to pre-cleared chromatin and incubate overnight at 4°C with rotation.

• Add 40μl Protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads sequentially with:

  • Low salt buffer (150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.0)

  • High salt buffer (500mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.0)

  • LiCl buffer (0.25M LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.0)

  • TE buffer (twice)

Elution and analysis:

• Elute DNA-protein complexes with elution buffer (1% SDS, 0.1M NaHCO₃) at 65°C.

• Reverse cross-links by incubating at 65°C overnight.

• Treat with RNase A and Proteinase K.

• Purify DNA using phenol-chloroform extraction or commercial kits.

• Analyze by qPCR, microarray, or next-generation sequencing.

Critical optimization parameters:

• Validate antibody specificity before ChIP experiments

• Include IgG control and input DNA samples

• Consider using dual cross-linking (formaldehyde plus DSG or EGS) for improved efficiency

• Test multiple antibody concentrations (3μg, 5μg, 10μg) to determine optimal conditions

How can OFUT37 antibodies be used for quantitative analysis of protein expression?

For precise quantitative analysis of OFUT37 protein expression, implement these methodological approaches:

Western blot-based quantification:

• Sample preparation standardization:

  • Extract proteins using a consistent buffer system (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors)

  • Determine protein concentration using BCA or Bradford assay

  • Load equal amounts (20-40μg) of total protein

• Standard curve development:

  • Generate a standard curve using purified recombinant OFUT37 protein (5-100ng range)

  • Process standards alongside experimental samples

  • Create a standard curve correlating band intensity with known protein amounts

• Detection optimization:

  • Use mid-range antibody dilution (1:1000) to ensure signal linearity

  • Select detection method with wide dynamic range (fluorescent secondary antibodies offer superior linearity compared to chemiluminescence)

  • Capture images using systems with 16-bit depth for expanded dynamic range

ELISA-based quantification:

• Sandwich ELISA development:

  • Coat plates with capture antibody (anti-OFUT37, 1-5μg/ml)

  • Add samples and standards (recombinant OFUT37)

  • Detect with a second OFUT37 antibody recognizing a different epitope

  • Measure absorbance and calculate concentrations from standard curve

• Competitive ELISA approach:

  • Coat plates with recombinant OFUT37 protein

  • Mix samples with a constant amount of detection antibody

  • Add mixture to plates and measure displacement of antibody binding

Flow cytometry for single-cell quantification:

• Fix and permeabilize cells/protoplasts

• Stain with OFUT37 antibody followed by fluorophore-conjugated secondary antibody

• Analyze expression at single-cell level using mean fluorescence intensity

Data normalization strategies:

• Normalize to total protein (Ponceau S staining)

• Use reference proteins (actin, tubulin) as loading controls

• Include spike-in controls of known concentration for absolute quantification

These approaches enable accurate quantification of OFUT37 expression across different experimental conditions and developmental stages.

What are emerging technologies for antibody-based detection that could enhance OFUT37 research?

Several cutting-edge technologies show promise for advancing OFUT37 antibody-based research:

Single-molecule detection technologies:

• Single-molecule pull-down (SiMPull): Combines principles of immunoprecipitation with single-molecule fluorescence imaging to detect OFUT37 interactions at the individual molecule level, revealing transient or low-abundance complexes often missed by traditional methods.

• Super-resolution microscopy techniques: Methods like STORM, PALM, and STED overcome the diffraction limit, enabling visualization of OFUT37 localization with 10-20nm resolution, potentially revealing previously undetectable microdomains or co-localization patterns.

Mass cytometry (CyTOF):
This technology combines flow cytometry with mass spectrometry, using antibodies labeled with rare earth metals instead of fluorophores. For OFUT37 research, CyTOF allows:

• Simultaneous detection of 40+ proteins without spectral overlap concerns

• Single-cell resolution of OFUT37 expression alongside numerous markers

• Integration with spatial information through Imaging Mass Cytometry

Proximity-dependent labeling:

• BioID or TurboID: Fusing biotin ligase to OFUT37 enables biotinylation of proximal proteins, which can be purified and identified by mass spectrometry. This approach maps the OFUT37 proximal proteome in living cells.

• APEX2 proximity labeling: Using an engineered peroxidase for proximity labeling provides temporal resolution of OFUT37 interactions with millisecond-scale reaction times.

Advanced microscopy platforms:

• Lattice light-sheet microscopy: Enables visualization of OFUT37 dynamics in living cells with minimal phototoxicity and exceptional temporal resolution.

• Correlative light and electron microscopy (CLEM): Combines immunofluorescence data of OFUT37 with ultrastructural context from electron microscopy.

Nanobody development:
Single-domain antibodies (nanobodies) derived from camelid antibodies offer several advantages for OFUT37 research:

• Smaller size enables better tissue penetration

• Improved access to sterically restricted epitopes

• Superior performance in super-resolution microscopy applications

These emerging technologies will significantly expand our understanding of OFUT37 biology by revealing previously inaccessible aspects of its localization, interactions, and dynamics.

How might OFUT37 antibodies contribute to understanding evolutionary conservation of O-fucosylation pathways across species?

OFUT37 antibodies can serve as powerful tools to explore the evolutionary conservation and divergence of O-fucosylation pathways:

Comparative immunodetection across plant lineages:
By testing OFUT37 antibodies against protein extracts from diverse plant species ranging from algae to angiosperms, researchers can:

• Construct a phylogenetic profile of OFUT37-like proteins

• Identify conserved domains through epitope mapping

• Correlate protein conservation with functional conservation

• Trace the evolutionary history of O-fucosylation machinery

Structural and functional domain mapping:
Using a panel of OFUT37 antibodies targeting different protein domains:

• Identify highly conserved regions suggesting functional importance

• Detect lineage-specific variations that may reflect adaptive changes

• Map catalytic domains through activity-blocking antibodies

• Correlate structural conservation with substrate specificity

Substrate conservation analysis:
Through immunoprecipitation coupled with mass spectrometry:

• Identify OFUT37 substrates across different species

• Compare O-fucosylation sites for evolutionary conservation

• Analyze adaptive shifts in substrate recognition

• Reconstruct the evolution of OFUT37-substrate networks

Comparative localization studies:
Immunolocalization of OFUT37 across diverse species can reveal:

• Conservation of subcellular targeting mechanisms

• Tissue-specific expression patterns

• Developmental regulation similarities and differences

• Correlations between localization and functional specialization

Integrative evolutionary analysis:
By combining antibody-based detection with genomic, transcriptomic, and phenotypic data:

• Construct comprehensive models of O-fucosylation pathway evolution

• Identify key evolutionary transitions and innovations

• Correlate molecular evolution with morphological complexity

• Discover lineage-specific adaptations in O-fucosylation pathways

This multifaceted approach using OFUT37 antibodies can provide unprecedented insights into how this important post-translational modification system has evolved across plant lineages and potentially reveal fundamental principles governing protein modification machinery evolution.

What are the key considerations for designing experiments with OFUT37 antibodies?

When designing experiments with OFUT37 antibodies, researchers should consider these critical factors to ensure reliable and reproducible results:

Antibody validation and selection:

• Verify antibody specificity using multiple approaches (Western blot, immunoprecipitation, knockout controls)

• Select antibodies appropriate for the intended application (fixed vs. live cells, denatured vs. native protein)

• Consider using antibody combinations targeting different epitopes for validation

• Document antibody characteristics (catalog number, lot, concentration) for reproducibility

Experimental design rigor:

• Include all necessary controls (negative, positive, isotype, blocking)

• Design experiments with appropriate statistical power

• Blind analysis where possible to prevent bias

• Standardize protocols for consistency across experiments

Technical considerations:

• Optimize antibody concentration for each application and sample type

• Determine appropriate incubation conditions (time, temperature, buffer composition)

• Validate antibody performance in your specific experimental system

• Consider potential cross-reactivity with related proteins

Data interpretation:

• Quantify results using appropriate methods

• Apply statistical analysis appropriate for the data type

• Consider biological relevance alongside statistical significance

• Present data with transparent reporting of experimental conditions

Integration with other approaches:

• Complement antibody-based methods with orthogonal techniques

• Consider genetic approaches (mutants, CRISPR/Cas9) alongside antibody studies

• Use computational predictions to guide experimental design

• Integrate findings with existing literature and databases