OFUT11 Antibody

What is OFUT11 Antibody and what protein does it target in Arabidopsis thaliana?

OFUT11 Antibody (product code CSB-PA787536XA01DOA) is a research-grade antibody that specifically targets the OFUT11 protein (UniProt accession: Q8GUM0) in Arabidopsis thaliana (Mouse-ear cress) . This antibody recognizes protein O-fucosyltransferase 11, which belongs to the GT65 family of glycosyltransferases and plays important roles in post-translational modification of target proteins through O-fucosylation. The antibody is typically supplied in 2ml/0.1ml volumes for research applications and has been validated for use in various immunoassay techniques in plant molecular biology research.

What are the primary applications of OFUT11 Antibody in plant molecular research?

OFUT11 Antibody serves multiple research applications in plant molecular biology, including:

• Immunolocalization studies to determine subcellular localization of OFUT11 protein

• Western blot analysis to quantify OFUT11 protein expression levels across different plant tissues or under various treatment conditions

• Immunoprecipitation assays to identify protein-protein interactions involving OFUT11

• Chromatin immunoprecipitation (ChIP) studies when investigating potential nuclear localization and function

• Flow cytometry applications for cell-specific expression analysis

When designing experiments, researchers should consider that antibody efficacy varies across these applications, with optimization required for each specific technique.

How should OFUT11 Antibody be stored and handled to maintain optimal activity?

For optimal preservation of OFUT11 Antibody activity, implement the following evidence-based storage and handling practices:

• Store antibody aliquots at -20°C for long-term storage to prevent freeze-thaw degradation

• For working solutions, maintain at 4°C for up to one month

• Avoid repeated freeze-thaw cycles; prepare single-use aliquots upon receipt

• When handling, minimize exposure to direct light, particularly for fluorophore-conjugated variants

• Use sterile techniques when preparing dilutions to prevent microbial contamination

• Document lot numbers and preparation dates to track antibody performance across experiments

These practices help maintain antibody specificity and sensitivity throughout the research timeline, ensuring consistent and reliable results.

What are the recommended dilution ratios for OFUT11 Antibody across different immunoassay techniques?

The optimal dilution ratios for OFUT11 Antibody vary by application technique and should be empirically determined for each new experimental system. Based on research protocols with plant antibodies of similar classes, the following ranges provide starting points:

When optimizing dilutions, begin with a titration series across the recommended range and evaluate signal-to-noise ratios to determine the optimal concentration for your specific experimental conditions.

What are the most effective protein extraction methods when working with OFUT11 Antibody in Arabidopsis tissues?

When extracting proteins for OFUT11 Antibody applications from Arabidopsis tissues, consider these methodological approaches:

• For general protein extraction, a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail provides effective solubilization while preserving antibody-epitope interactions.

• When isolating membrane-associated proteins (as OFUT11 may localize to the endoplasmic reticulum), incorporate 0.1% SDS or 6M urea into the extraction buffer to improve solubilization while maintaining epitope integrity.

• For nuclear protein extraction when investigating potential nuclear functions, use specialized nuclear extraction kits or protocols with high-salt buffers (containing 420mM NaCl) following initial cytoplasmic fraction removal.

• Always include fresh protease inhibitors and perform extractions at 4°C to minimize protein degradation which could affect antibody recognition.

• Consider tissue-specific optimization as protein content varies between roots, leaves, flowers, and siliques in Arabidopsis thaliana.

How can researchers address non-specific binding when using OFUT11 Antibody in Western blot applications?

When encountering non-specific binding with OFUT11 Antibody in Western blot applications, implement these evidence-based troubleshooting strategies:

• Optimize blocking conditions by testing different blocking agents (5% non-fat dry milk, 3-5% BSA, or commercial blocking buffers) and extending blocking time to 2 hours at room temperature or overnight at 4°C.

• Adjust antibody dilution and incubation conditions - increase dilution incrementally (e.g., from 1:1000 to 1:2000) and conduct incubations at 4°C overnight rather than at room temperature.

• Include additional washing steps with increasing stringency by adding 0.1-0.3% Tween-20 to TBST/PBST washing buffers, and extend washing durations to 10 minutes per wash with at least 5 wash cycles.

• Pre-absorb the antibody with Arabidopsis protein extract from ofut11 knockout mutants or tissues with low OFUT11 expression to remove antibodies that might recognize non-specific epitopes.

• Validate results with appropriate controls, including pre-immune serum controls, secondary antibody-only controls, and competition assays with purified antigen when available.

What cross-reactivity considerations should researchers be aware of when working with OFUT11 Antibody?

When designing experiments with OFUT11 Antibody, researchers should address these cross-reactivity considerations:

• The antibody may cross-react with orthologous O-fucosyltransferases in Arabidopsis, particularly OFUT12 and OFUT13, which share sequence homology with OFUT11. Verify specificity using knockout mutant controls or by performing parallel experiments with antibodies specific to these related proteins.

• For cross-species applications, note that OFUT11 homologs exist in other plant species with varying degrees of conservation. While the antibody is raised against Arabidopsis thaliana OFUT11 (UniProt: Q8GUM0) , cross-reactivity with orthologs from closely related Brassicaceae species is possible but requires validation.

• Sequence alignment analysis indicates higher homology in the catalytic domain versus regulatory regions of O-fucosyltransferases across species. Consider the antibody's epitope location when evaluating potential cross-reactivity.

• When working with tissue samples containing microorganisms or symbionts, be aware that some bacterial and fungal glycosyltransferases may share structural similarities with plant O-fucosyltransferases, potentially causing unexpected cross-reactivity.

How can OFUT11 Antibody be effectively utilized in co-immunoprecipitation studies to identify interaction partners?

For effective co-immunoprecipitation (Co-IP) studies with OFUT11 Antibody to identify protein interaction partners:

• Select appropriate lysis conditions that preserve protein-protein interactions while effectively solubilizing OFUT11. A buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 0.5% NP-40, 1mM EDTA, 10% glycerol with protease inhibitors is a recommended starting point.

• For the immunoprecipitation procedure:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with OFUT11 Antibody at 1:50-1:100 dilution for 4 hours or overnight at 4°C

  • Add pre-washed Protein A/G beads and incubate for an additional 2-3 hours

  • Perform stringent washing (at least 5 times) with lysis buffer containing reduced detergent

• Include these critical controls:

  • IgG-matched isotype control to identify non-specific interactions

  • Input sample (5-10% of lysate) to verify protein presence in starting material

  • Reciprocal Co-IP with antibodies against suspected interaction partners

  • When available, OFUT11 knockout or knockdown samples as negative controls

• For detection of interaction partners, consider mass spectrometry analysis following silver staining of separated proteins, or Western blot analysis if specific partners are suspected.

• Validate identified interactions through secondary methods such as bimolecular fluorescence complementation (BiFC), FRET analysis, or in vitro binding assays.

What considerations are important when using OFUT11 Antibody for immunolocalization studies in plant tissues?

When conducting immunolocalization studies with OFUT11 Antibody in plant tissues, consider these advanced methodological factors:

• Fixation protocol optimization is critical:

  • For paraffin-embedded sections, 4% paraformaldehyde fixation for 12-16 hours is generally effective

  • For cryosections, shorter fixation (1-2 hours) in 2% paraformaldehyde may better preserve epitope accessibility

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 8.0, enzymatic retrieval) to determine optimal epitope exposure

• When examining potentially low-abundance OFUT11 expression:

  • Implement tyramide signal amplification to enhance detection sensitivity

  • Use confocal microscopy with spectral unmixing to distinguish specific signal from autofluorescence

  • Consider dual labeling with organelle markers (ER, Golgi, plasma membrane) to precisely define subcellular localization

• For co-localization studies with potential interaction partners:

  • Select fluorophores with minimal spectral overlap

  • Include appropriate single-label controls

  • Quantify co-localization using statistical methods (Pearson's correlation coefficient, Mander's overlap coefficient)

  • Apply super-resolution techniques (STED, PALM, STORM) for precise spatial relationship analysis

How should researchers quantitatively analyze Western blot data when studying OFUT11 expression across different developmental stages or stress conditions?

For rigorous quantitative analysis of OFUT11 expression using Western blot data:

• Implement standardized densitometric analysis:

  • Use software packages that allow background subtraction and normalization (ImageJ, Image Lab, etc.)

  • Define analysis boundaries consistently across samples

  • Apply local background correction for each lane individually

• Proper normalization is essential:

  • Utilize loading controls appropriate for your experimental conditions (GAPDH, actin, or tubulin for general analysis; compartment-specific controls like BiP for ER-localized proteins)

  • Verify linearity of loading control signal across your sample concentration range

  • Calculate OFUT11 signal relative to loading control for each sample

• For time-course or treatment comparisons:

  • Express data as fold-change relative to control conditions

  • Perform statistical analysis across biological replicates (minimum n=3)

  • Apply appropriate statistical tests (ANOVA with post-hoc tests for multiple comparisons)

  • Generate error bars representing standard deviation or standard error

• Address common quantification challenges:

  • For saturated signals, prepare a dilution series to ensure measurements fall within the linear detection range

  • When analyzing post-translational modifications, calculate modified-to-unmodified protein ratios

  • For samples with widely varying expression levels, consider multiple exposure times or dilution series

What methodological approaches help distinguish between specific and non-specific signals when using OFUT11 Antibody in immunofluorescence studies?

To reliably distinguish between specific and non-specific signals in OFUT11 Antibody immunofluorescence studies:

• Implement comprehensive control experiments:

  • Secondary antibody-only controls to identify background fluorescence

  • Pre-immune serum controls to establish baseline non-specific binding

  • Peptide competition assays where the antibody is pre-incubated with excess antigen peptide

  • When available, tissue from ofut11 knockout or knockdown plants as negative controls

  • Known OFUT11-overexpressing tissues or transgenic lines as positive controls

• Apply advanced imaging techniques:

  • Spectral imaging to separate specific signal from autofluorescence, particularly important in plant tissues with chlorophyll and cell wall components

  • Lambda scanning to generate spectral fingerprints of true signal versus autofluorescence

  • Time-resolved imaging for fluorophores with distinct fluorescence lifetimes

• Optimize image acquisition parameters:

  • Determine threshold settings using control samples

  • Maintain identical acquisition parameters across all experimental conditions

  • Capture z-stacks to ensure complete signal detection throughout the tissue

• For quantitative analysis:

  • Define signal intensity thresholds based on control samples

  • Measure signal-to-noise ratios across different experimental conditions

  • Apply colocalization analysis with known markers of expected OFUT11 localization

  • Use deconvolution algorithms to improve signal resolution and specificity

How can OFUT11 Antibody be effectively utilized in ChIP-seq studies to investigate potential roles in transcriptional regulation?

For researchers exploring potential nuclear functions of OFUT11 through ChIP-seq applications:

• Protocol optimization for plant ChIP-seq with OFUT11 Antibody:

  • Test crosslinking conditions (1% formaldehyde for 10-15 minutes is standard, but optimize duration)

  • Evaluate sonication parameters to achieve 200-500bp chromatin fragments

  • Determine optimal antibody concentration through titration experiments

  • Include appropriate input controls and IgG controls

• Critical validation steps:

  • Perform Western blot analysis of nuclear fractions to confirm OFUT11 presence

  • Conduct ChIP-qPCR validation of enriched regions before sequencing

  • Include biological replicates (minimum n=3) to identify reproducible binding sites

  • When possible, compare results with ChIP-seq data using tagged OFUT11 constructs

• Bioinformatic analysis considerations:

  • Use peak calling algorithms suited for transcription factor ChIP-seq

  • Perform motif enrichment analysis to identify potential DNA binding motifs

  • Compare binding sites with transcriptome data to correlate binding with gene expression

  • Integrate with datasets for histone modifications or other transcription factors

• Functional validation of identified targets:

  • Conduct reporter gene assays for selected targets

  • Perform genetic studies using ofut11 mutants to verify regulatory relationships

  • Investigate protein-protein interactions with known transcriptional regulators

What emerging technologies are enhancing the applications of OFUT11 Antibody in plant glycobiology research?

Recent technological advances are expanding the application scope of OFUT11 Antibody in plant glycobiology:

• Proximity labeling methodologies:

  • APEX2 or BioID fusion proteins combined with OFUT11 Antibody immunoprecipitation enable identification of proximal proteins in the native cellular environment

  • These approaches help map the OFUT11 interactome with spatial resolution

  • Integration with mass spectrometry allows unbiased identification of both protein partners and potential substrates

• Advanced glycoproteomics applications:

  • Combining OFUT11 Antibody immunoprecipitation with glycan-specific labeling techniques

  • Sequential enrichment strategies using lectin affinity followed by OFUT11 immunoprecipitation

  • Mass spectrometry workflows optimized for O-fucosylated peptide identification

• Super-resolution microscopy integration:

  • STORM and PALM microscopy combined with OFUT11 Antibody provides nanoscale resolution of localization

  • Correlative light and electron microscopy (CLEM) allows precise ultrastructural contextualization

  • Expansion microscopy protocols adapted for plant tissues enable improved spatial resolution with standard confocal microscopy

• CRISPR-based approaches:

  • Epitope tagging of endogenous OFUT11 at the genomic level

  • Generation of knock-in reporter lines that maintain native regulation

  • Development of degron-tagged OFUT11 for controlled protein depletion studies

How does the performance of OFUT11 Antibody compare with antibodies targeting other O-fucosyltransferases in plant research?

When selecting between antibodies targeting different O-fucosyltransferases for plant research:

• Specificity comparison across the OFUT family:

  • OFUT11 Antibody typically shows higher specificity compared to antibodies targeting OFUT15 and OFUT20 due to lower sequence homology in immunogenic regions

  • Cross-reactivity profiles differ significantly, with OFUT11 Antibody showing minimal cross-reactivity with OFUT29 but potential recognition of OFUT11 homologs in closely related species

• Performance across techniques:

  • OFUT11 Antibody demonstrates superior performance in Western blot applications compared to other OFUT antibodies

  • For immunolocalization, OFUT20 Antibody may provide better signal-to-noise ratios in certain tissue types

  • In immunoprecipitation applications, efficiency varies based on protein abundance and complex stability

• Experimental validation metrics:

  • Sensitivity: OFUT11 Antibody typically detects target protein at concentrations as low as 0.1 ng/μl

  • Specificity: Confirmable through knockout controls and peptide competition assays

  • Reproducibility: Batch-to-batch variation is minimized through standardized production protocols

• Application-specific recommendations:

  • For multi-protein complex studies, consider using a combination of antibodies against different OFUT family members

  • When studying potential redundant functions, validate specificity of each antibody using recombinant proteins

What methodological adaptations are required when using OFUT11 Antibody in non-Arabidopsis plant species or heterologous expression systems?

When extending OFUT11 Antibody applications beyond Arabidopsis thaliana:

• For other plant species:

  • Perform sequence alignment analysis to assess epitope conservation

  • In closely related Brassicaceae species, the antibody typically performs well with standard protocols

  • For more distant species, increase antibody concentration by 1.5-2 fold and extend incubation times

  • Validate specificity with heterologous expression of Arabidopsis OFUT11 as a positive control

• When using in crop species:

  • For monocots (rice, wheat, maize), protein extraction protocols require modification to address higher polysaccharide and phenolic compound content

  • Include additional detergents (0.1% SDS) and antioxidants (1-2 mM DTT) in extraction buffers

  • Extend blocking times to minimize background in Western blot applications

  • Consider using tissue-specific extraction protocols for specialized tissues like endosperm or pollen

• For heterologous expression systems:

  • In yeast systems, modify lysis buffers to account for different membrane compositions

  • For insect cell expression, validate glycosylation patterns that might affect epitope recognition

  • In bacterial expression systems, confirm proper protein folding which may impact epitope structure

• Protocol optimization considerations:

  • Test a gradient of antibody concentrations to determine optimal dilution for each system

  • Modify fixation protocols for immunohistochemistry based on tissue permeability differences

  • Adjust antigen retrieval methods according to species-specific cell wall compositions