OTUD3 Antibody, FITC conjugated

What is OTUD3 and why is it an important research target?

OTUD3 (OTU domain-containing protein 3) is a deubiquitinating enzyme (DUB) that hydrolyzes 'Lys-6'- and 'Lys-11'-linked polyubiquitin chains as well as heterotypic (mixed and branched) and homotypic chains . It has emerged as a critical protein with context-dependent roles in cancer biology and neurological disorders. OTUD3 functions as a tumor suppressor in breast, colon, liver and cervical cancers by stabilizing the tumor suppressor PTEN, while paradoxically promoting lung tumorigenesis through stabilizing GRP78 . Recent research has also implicated OTUD3 in endoplasmic reticulum stress and dopaminergic neuron neurodegeneration . Its diverse and tissue-specific roles make OTUD3 an important target for understanding ubiquitin-mediated protein regulation.

What are the technical specifications of the OTUD3 Antibody, FITC conjugated?

The OTUD3 Antibody, FITC conjugated is a polyclonal antibody derived from rabbit hosts that specifically recognizes human OTUD3. The immunogen used for antibody production is recombinant human OTU domain-containing protein 3 protein (amino acids 108-398). This antibody is supplied in a liquid form with a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. It has been validated for ELISA applications and is isotype IgG . The FITC (fluorescein isothiocyanate) conjugation enables direct fluorescent detection without the need for secondary antibodies.

How should the OTUD3 Antibody, FITC conjugated be stored to maintain optimal activity?

For optimal preservation of antibody activity, the OTUD3 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can significantly degrade antibody performance . When working with the antibody, it's recommended to aliquot the stock solution into smaller volumes before freezing to minimize freeze-thaw cycles. FITC conjugates are light-sensitive, so storage and handling should be conducted under reduced light conditions to prevent photobleaching of the fluorophore.

What are the optimal conditions for using OTUD3 Antibody, FITC conjugated in immunofluorescence experiments?

When designing immunofluorescence experiments with OTUD3 Antibody, FITC conjugated, researchers should consider:

• Fixation method: Paraformaldehyde (4%) for 15-20 minutes at room temperature typically works well for preserving OTUD3 without compromising epitope recognition.

• Permeabilization: Use 0.1-0.3% Triton X-100 for 5-10 minutes to allow antibody access to intracellular OTUD3.

• Blocking: 5-10% normal serum (matched to the host species of secondary antibodies) with 1% BSA for 1 hour helps reduce background.

• Antibody dilution: Start with 1:50 to 1:200 dilutions and optimize based on signal-to-noise ratio.

• FITC considerations: FITC has an excitation maximum at ~495 nm and emission maximum at ~519 nm. Ensure your microscopy setup can detect this wavelength range.

• Controls: Include a no-primary antibody control and, if possible, OTUD3 knockout cells as a negative control .

• Counterstaining: DAPI nuclear stain (blue) provides good contrast with the green FITC signal.

Since OTUD3 has been reported to shuttle between nuclear and cytoplasmic compartments and associate with microtubules , careful image analysis should be performed to accurately determine subcellular localization.

How can I validate the specificity of OTUD3 Antibody, FITC conjugated in my experimental system?

Validating antibody specificity is critical for reliable research results. For OTUD3 Antibody, FITC conjugated, consider these validation approaches:

• Western blot analysis: Though this antibody is primarily validated for ELISA, running parallel samples with a validated OTUD3 antibody can confirm target recognition. OTUD3 protein should appear at approximately 48 kDa.

• OTUD3 knockout/knockdown controls: OTUD3 knockout cell lines have been generated and reported in the literature . These provide excellent negative controls to confirm antibody specificity.

• Peptide competition assay: Pre-incubating the antibody with excess immunizing peptide (OTUD3 amino acids 108-398) should abolish specific staining.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is pulling down OTUD3 rather than cross-reacting with other proteins.

• Sibling antibody comparison: Compare results with other validated OTUD3 antibodies targeting different epitopes.

• Signal correlation with known OTUD3 expression patterns: The antibody signal should be stronger in cells known to express higher levels of OTUD3, such as certain lung cancer cell lines (H1299, A549) .

What are the most appropriate experimental controls when using this antibody for studying OTUD3's role in cancer models?

When investigating OTUD3's role in cancer using this antibody, the following controls are essential:

• Cell line panel controls: Include both OTUD3-dependent cancer cells (such as lung cancer cell lines H1299, A549, H460, and H1650) and cancer types where OTUD3 plays a tumor-suppressive role (breast cancer lines MCF7, MDA-MB-231) .

• Normal tissue controls: Compare antibody staining patterns between cancer cells and corresponding normal cells (such as human pulmonary alveolar epithelial cells (HPAEpiC) for lung cancer studies) .

• Genetic controls:

  • OTUD3 knockdown/knockout cells

  • OTUD3 overexpression cells

  • Rescue experiments with wild-type vs. mutant OTUD3

• Treatment controls:

  • Proteasome inhibitors (MG132) which should increase OTUD3 substrate levels

  • OTUD3 inhibitors such as OTUDin3

  • Protein synthesis inhibitor cycloheximide for protein stability studies

• Co-localization controls: Given OTUD3's interactions with GRP78 and PTEN, co-staining with antibodies against these proteins can validate functional relationships .

How can OTUD3 Antibody, FITC conjugated be used to study the differential roles of OTUD3 in cancer progression?

The OTUD3 Antibody, FITC conjugated can be strategically employed to investigate OTUD3's dual role in cancer:

• Comparative expression analysis: Use flow cytometry to quantitatively compare OTUD3 expression levels across multiple cancer cell lines (breast, colon, liver, cervical, and lung cancers) where OTUD3 has opposing functions .

• Substrate co-localization studies: Perform dual immunofluorescence microscopy to visualize co-localization of OTUD3 with its different substrates:

  • OTUD3 and PTEN in breast cancer cells (tumor-suppressive context)

  • OTUD3 and GRP78 in lung cancer cells (oncogenic context)

• Temporal dynamics analysis: Use live-cell imaging with the FITC-conjugated antibody in permeabilized cells to track OTUD3 localization changes during cancer progression or in response to treatments.

• High-content screening applications: Employ the antibody in high-throughput immunofluorescence screens to identify compounds that modulate OTUD3 expression or localization differently in various cancer contexts.

• Patient-derived xenograft (PDX) models: Use the antibody to track OTUD3 expression and localization in different tumor microenvironments using tissue sections from PDX models of various cancer types.

This approach can help elucidate the molecular mechanisms underlying OTUD3's context-dependent functions in different cancer types .

What experimental approaches can be used to investigate the relationship between OTUD3 and CHIP using this antibody?

To study the regulatory relationship between OTUD3 and CHIP (Carboxyl terminus of Hsc70-interacting protein), researchers can implement these approaches using the OTUD3 Antibody, FITC conjugated:

• Co-immunoprecipitation with dual visualization:

  • Precipitate with anti-CHIP antibody

  • Detect OTUD3 using the FITC-conjugated antibody via fluorescence imaging of the precipitated complex

  • Use differentially labeled antibodies to simultaneously visualize both proteins

• Proximity ligation assay (PLA):

  • Combine OTUD3 Antibody, FITC conjugated with anti-CHIP antibody

  • Use proximity probes to detect protein-protein interactions in situ

  • Quantify interaction signals across different experimental conditions

• CHIP manipulation experiments:

  • Track OTUD3 levels using flow cytometry with the FITC-conjugated antibody in:

    • CHIP-knockdown cells (should show increased OTUD3 levels)

    • CHIP-overexpressing cells (should show decreased OTUD3 levels)

    • Cells expressing CHIP mutants (H260Q or K30A) that lack E3 ligase activity

• Ubiquitination assays:

  • Monitor OTUD3 ubiquitination status after CHIP manipulation

  • Correlate with OTUD3 protein levels detected by fluorescence intensity

• Domain mapping:

  • Use truncated OTUD3 constructs to identify which domains are necessary for CHIP interaction

  • Monitor co-localization using the FITC-conjugated antibody and an anti-CHIP antibody

These approaches would help validate that CHIP acts as a ubiquitin ligase for OTUD3, promoting its polyubiquitylation and subsequent degradation, as suggested by previous research .

How can I use this antibody to evaluate the efficacy of OTUD3 inhibitors in cellular models?

To assess OTUD3 inhibitor efficacy using the OTUD3 Antibody, FITC conjugated:

• Target engagement evaluation:

  • Implement cellular thermal shift assays (CETSA) with FITC fluorescence readout

  • Monitor thermal stability changes of OTUD3 upon inhibitor binding

  • Quantify shifts in melting temperature as indicators of direct inhibitor binding

• Functional consequences assessment:

  • Use flow cytometry to quantify changes in OTUD3 substrate levels (GRP78) upon inhibitor treatment

  • Monitor cell death markers simultaneously with OTUD3 levels

  • Create dose-response curves correlating inhibitor concentration with:

    • OTUD3 activity (measured by substrate levels)

    • Cellular phenotypes (apoptosis, cell cycle arrest)

• High-content microscopy applications:

  • Track subcellular localization changes of OTUD3 following inhibitor treatment

  • Quantify co-localization coefficients between OTUD3 and its substrates

  • Monitor morphological changes in treated cells

• Combination therapy screening:

  • Use the antibody to assess OTUD3 inhibition in combination with standard chemotherapeutics

  • Identify synergistic drug combinations through automated image analysis

• Resistance mechanism investigation:

  • Monitor OTUD3 expression changes in cell populations developing resistance to inhibitors

  • Track compensatory mechanisms through co-staining with antibodies against related DUBs

These approaches would be particularly valuable for evaluating novel OTUD3 inhibitors like OTUDin3, which has shown promise in treating non-small cell lung cancer by disrupting the deubiquitinating activity of OTUD3 toward GRP78 .

What strategies can resolve weak or absent signals when using OTUD3 Antibody, FITC conjugated?

When encountering weak or absent signals with the OTUD3 Antibody, FITC conjugated, consider these methodological optimizations:

• Sample preparation improvements:

  • Optimize fixation time (try 10, 15, or 20 minutes with 4% PFA)

  • Test different permeabilization reagents (0.1% Triton X-100, 0.1% Saponin, or 100% methanol)

  • Verify that your cell type expresses detectable levels of OTUD3

• Antibody incubation optimization:

  • Increase antibody concentration (try 1:25 dilution)

  • Extend incubation time (overnight at 4°C)

  • Add 0.1% BSA to antibody dilution buffer to prevent non-specific binding

• Signal enhancement techniques:

  • Implement tyramide signal amplification (TSA) for FITC signal boosting

  • Use anti-FITC secondary antibodies conjugated to brighter fluorophores

  • Apply photobleaching protectants in mounting media

• Technical considerations:

  • Ensure microscope settings are optimized for FITC detection

  • Check filter sets for proper excitation/emission wavelengths

  • Verify that image acquisition settings are sensitive enough

• Positive control implementation:

  • Include cells with confirmed high OTUD3 expression (H1299 lung cancer cells)

  • Process a sample with a validated non-conjugated OTUD3 antibody in parallel

If signal remains problematic, consider whether OTUD3 might be degraded in your specific experimental conditions, as CHIP-mediated degradation of OTUD3 has been documented .

How can I minimize background fluorescence when using this antibody in tissue sections?

To reduce background fluorescence when using OTUD3 Antibody, FITC conjugated in tissue sections:

• Tissue preparation optimization:

  • Perfuse-fix animals when possible for cleaner tissue preparation

  • Use freshly prepared paraformaldehyde and optimal fixation times

  • Consider antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Blocking enhancement:

  • Implement dual blocking strategy:

    • 5-10% normal serum (from species unrelated to the primary antibody)

    • 0.1-0.3% Triton X-100

    • 1% BSA

  • Add 0.1-0.3% glycine to quench aldehyde groups from fixation

  • Consider mouse-on-mouse blocking reagents for mouse tissues

• Autofluorescence reduction:

  • Treat sections with 0.1-1% sodium borohydride for 5 minutes before antibody incubation

  • Use Sudan Black B (0.1-0.3% in 70% ethanol) after antibody incubation

  • Apply commercial autofluorescence quenchers specifically designed for FITC wavelengths

• Washing optimization:

  • Extend wash times (5-6 washes, 10 minutes each)

  • Add 0.05% Tween-20 to wash buffers

  • Perform washes on an orbital shaker for better removal of unbound antibody

• Imaging considerations:

  • Use confocal microscopy to reduce out-of-focus fluorescence

  • Apply spectral unmixing to separate FITC signal from autofluorescence

  • Consider tissue-specific autofluorescence fingerprints (e.g., lipofuscin in aged tissues)

Careful titration of the antibody concentration is particularly important for tissue sections to find the optimal balance between specific signal and background.

What experimental modifications are needed when using this antibody to study OTUD3's role in endoplasmic reticulum stress?

When investigating OTUD3's role in endoplasmic reticulum (ER) stress using the OTUD3 Antibody, FITC conjugated, implement these methodological adaptations:

• ER stress induction protocols:

  • Tunicamycin (0.5-5 μg/ml, 6-24 hours)

  • Thapsigargin (0.1-1 μM, 6-24 hours)

  • DTT (1-2 mM, 1-4 hours)

  • Monitor ER stress markers (BiP/GRP78, XBP1s, ATF6, phospho-PERK) in parallel with OTUD3

• Co-localization with ER markers:

  • Co-stain with ER markers (calnexin, PDI, or KDEL-containing proteins)

  • Use spectrally distinct fluorophores that don't overlap with FITC

  • Implement super-resolution microscopy for precise localization

• Substrate interaction analysis:

  • Monitor OTUD3-Fortilin interactions during ER stress

  • Track IRE1α activation status in relation to OTUD3 levels

  • Assess XBP1 splicing as a downstream readout of IRE1α activity

• Time-course experiments:

  • Track OTUD3 levels and localization at multiple timepoints after ER stress induction

  • Correlate OTUD3 dynamics with the progression of the unfolded protein response

• Neuronal model considerations:

  • For studies in dopaminergic neurons, implement:

    • Specific neuronal markers (TH, DAT) for co-labeling

    • Careful fixation protocols to preserve neuronal morphology

    • Analysis of neurodegeneration markers alongside OTUD3

• Inhibitor studies:

  • Compare ER stress responses in the presence of OTUD3 inhibitors

  • Monitor downstream consequences on cell viability

These approaches will help elucidate OTUD3's role in regulating Fortilin and IRE1α signaling during ER stress, particularly in neuronal contexts where OTUD3 has been implicated in neurodegeneration .

How should I interpret changes in OTUD3 localization patterns in different cellular contexts?

When analyzing OTUD3 localization patterns using the FITC-conjugated antibody, consider these interpretation guidelines:

• Subcellular distribution profiles:

  • Cytoplasmic predominance: Typical pattern in many cell types

  • Nuclear localization: May indicate activation of specific signaling pathways

  • Perinuclear accumulation: Often associated with ER stress responses

  • Microtubule association: Recently discovered function of OTUD3

• Context-dependent interpretation:

  • Cancer context: Compare localization between:

    • Tissues where OTUD3 is tumor-suppressive (breast, colon)

    • Tissues where OTUD3 is oncogenic (lung)

  • Neuronal context: Assess correlation between OTUD3 localization and:

    • ER stress markers

    • Neurodegeneration indicators

• Quantitative analysis approaches:

  • Nuclear/cytoplasmic ratio calculations using image analysis software

  • Co-localization coefficients with organelle markers (Pearson's, Mander's)

  • Intensity distribution profiles across cellular compartments

• Dynamic changes interpretation:

  • Rapid translocation may indicate stress response

  • Gradual redistribution might reflect adaptive cellular changes

  • Consider cell cycle phase influences on localization

• Correlation with functional outcomes:

  • Relate localization changes to:

    • Substrate levels (GRP78, PTEN, Fortilin)

    • Cellular phenotypes (proliferation, migration, apoptosis)

    • Treatment responses

Remember that OTUD3 contains both nuclear export signals (NES) and putative nuclear localization signals (NLS) , suggesting regulated nuclear-cytoplasmic shuttling that may be functionally significant.

What statistical approaches are most appropriate for quantifying OTUD3 expression levels across experimental conditions?

For robust statistical analysis of OTUD3 expression data obtained using the FITC-conjugated antibody:

• Flow cytometry data analysis:

  • Mean fluorescence intensity (MFI) comparisons:

    • Paired t-tests for before/after treatment

    • ANOVA with post-hoc tests for multiple condition comparisons

  • Population distribution analysis:

    • Kolmogorov-Smirnov test for comparing distribution shapes

    • Subpopulation identification via clustering algorithms

• Immunofluorescence microscopy quantification:

  • Single-cell analysis approaches:

    • Measure integrated density values from individual cells (n≥100 per condition)

    • Use hierarchical linear models to account for cell-to-cell variability

  • Region-of-interest (ROI) strategies:

    • Define consistent ROIs across samples

    • Implement background subtraction methods before comparison

• Accounting for confounding variables:

  • Normalize to:

    • Cell size

    • Total protein content

    • Housekeeping protein expression

  • Use ANCOVA when covariates might influence expression

• Multiple testing correction:

  • Benjamini-Hochberg procedure for controlling false discovery rate

  • Bonferroni correction for stringent family-wise error rate control

• Data visualization approaches:

  • Violin plots to show distribution characteristics

  • Forest plots for meta-analysis across experimental replicates

  • Heat maps for correlation analysis with multiple markers

For longitudinal studies tracking OTUD3 changes over time, consider repeated measures ANOVA or mixed-effects models to account for within-subject correlations.

How can I reconcile contradictory findings regarding OTUD3 function between different experimental models?

When encountering contradictory results regarding OTUD3 function across different experimental systems:

• Context-dependent functions assessment:

  • Systematically document tissue/cell type differences:

    • OTUD3 is tumor-suppressive in breast, colon, liver, cervical cancer

    • OTUD3 is oncogenic in lung cancer

    • OTUD3 may have protective roles in neuronal cells

  • Map substrate availability across systems:

    • PTEN stabilization dominates in some contexts

    • GRP78 stabilization predominates in others

    • Fortilin regulation matters in neuronal contexts

• Methodological reconciliation strategies:

  • Standardize experimental conditions across models:

    • Use identical antibody concentrations and protocols

    • Implement consistent quantification methods

    • Apply the same statistical analyses

  • Perform side-by-side comparisons in a single laboratory

• Molecular mechanism integration:

  • Map OTUD3 interaction networks across cell types

  • Identify tissue-specific co-factors that might redirect OTUD3 function

  • Consider post-translational modifications that might differ between systems

• Genetic background considerations:

  • Account for variations in:

    • OTUD3 expression levels

    • Mutation status of key interaction partners

    • Polymorphisms in OTUD3 regulatory regions

• Proposed unifying models:

  • Develop conceptual frameworks that accommodate context-dependent functions

  • Consider threshold effects where OTUD3 function changes based on expression level

  • Map signaling pathway intersections that might dictate functional outcomes