DTX3 Antibody, FITC conjugated

What is DTX3 and why is it an important research target?

DTX3 (Deltex Homolog 3) is a 347 amino acid protein with a calculated molecular weight of 38 kDa that contains one RING-type zinc finger domain and belongs to the Deltex family. It functions as a regulator of Notch signaling, which is involved in cell-cell communications that regulate a broad spectrum of cell-fate determinations . The importance of DTX3 in research stems from its role in critical cellular pathways and potential implications in various biological processes. As a component of the Notch signaling pathway, it influences developmental processes and cellular differentiation, making it relevant for studies in developmental biology, cancer research, and cell signaling.

What is the molecular structure and cellular localization of DTX3?

DTX3, also known as Deltex3 or RING finger protein 154, has a predicted molecular weight of 38 kDa and consists of 347 amino acids. Its structure includes a RING-type zinc finger domain characteristic of the Deltex family . DTX3 contains specific epitopes that can be targeted by antibodies, particularly in the regions spanning amino acids 90-347, 201-301, and other specific regions as indicated in various antibody products . The protein is primarily localized in cellular compartments involved in Notch signaling pathways, where it participates in protein-protein interactions through its RING domain, potentially involved in ubiquitination processes.

What is FITC conjugation and how does it enable antibody-based detection?

FITC (Fluorescein isothiocyanate) conjugation is the chemical process of covalently linking FITC molecules to antibodies, typically through reaction with primary amines (lysines) on the antibody structure . This conjugation creates a fluorescent-labeled antibody that can be detected using fluorescence-based techniques. When exposed to light at its excitation wavelength (approximately 495 nm), FITC emits green fluorescence at around 519 nm , allowing visualization of the antibody-antigen binding in techniques such as flow cytometry, immunofluorescence microscopy, and immunohistochemistry. The conjugation enables direct detection without requiring secondary antibodies, streamlining experimental workflows while maintaining the antibody's target specificity.

What is the optimal protocol for conjugating FITC to DTX3 antibodies?

The optimal protocol for FITC conjugation to DTX3 antibodies involves several critical steps:

Preparation Phase:

• Ensure antibody concentration is at least 2 mg/ml in a compatible buffer

• Prepare fresh FITC solution (unstable once solubilized)

• Adjust reaction conditions to pH 9.5 with carbonate buffer

Conjugation Procedure:

• Add FITC to antibody solution at various molar ratios (typically aiming for 3-6 FITC molecules per antibody)

• Incubate at room temperature for 30-60 minutes with gentle agitation

• Stop reaction with ammonium chloride or glycine

Purification:

• Remove unconjugated FITC using gel filtration chromatography

• Separate optimally labeled antibodies using gradient DEAE Sephadex chromatography

• Collect fractions and analyze for fluorescein/protein (F/P) ratio

Research indicates that maximal labeling is obtained at room temperature, pH 9.5, and an initial protein concentration of 25 mg/ml . Multiple parallel reactions with different FITC-to-antibody ratios should be performed to determine the optimal conjugation conditions for each specific antibody .

How can I determine the fluorescein/protein (F/P) ratio of my FITC-conjugated DTX3 antibody?

Determining the fluorescein/protein (F/P) ratio is essential for characterizing FITC-conjugated antibodies and predicting their performance in experiments. This can be accomplished through spectrophotometric analysis:

Spectrophotometric Method:

• Measure absorbance of the conjugate at 280 nm (A₂₈₀) and 495 nm (A₄₉₅)

• Calculate the F/P ratio using the formula:
  F/P ratio = [A₄₉₅ × dilution factor] / [A₂₈₀ - (0.35 × A₄₉₅)] × 2.87

Interpretation:

• Optimal F/P ratio typically ranges between 3-6 FITC molecules per antibody

• Higher ratios (>6) may cause internal quenching and reduced brightness

• Lower ratios (<3) may result in insufficient signal intensity

Research has demonstrated that the FITC-labeling index is negatively correlated with binding affinity for target antigens . Therefore, it's crucial to balance fluorescence intensity with maintained antigen recognition. Gradient DEAE Sephadex chromatography can be used to separate antibodies with different F/P ratios, allowing selection of those with optimal characteristics .

What effect does FITC conjugation have on DTX3 antibody binding affinity?

FITC conjugation can significantly impact the binding affinity of DTX3 antibodies to their target antigens, with several important considerations:

Impact on Binding Properties:

Research has demonstrated that the FITC-labeling index in antibodies is negatively correlated with binding affinity for target antigens . This occurs because FITC molecules conjugate to lysine residues, some of which may be located within or near the antigen-binding sites of the antibody. Immunohistochemically, antibodies with higher labeling indices tend to be more sensitive but are also more likely to yield non-specific staining .

For DTX3 antibodies specifically, it's recommended to carefully select FITC-labeled antibodies from several differently labeled preparations to minimize the decrease in binding affinity while achieving appropriate sensitivity for the intended application .

What are the optimal applications for FITC-conjugated DTX3 antibodies in research?

FITC-conjugated DTX3 antibodies are versatile tools applicable across multiple research techniques, with each application having specific advantages:

Flow Cytometry:

• Optimal for quantifying DTX3 expression in cell populations

• Allows multi-parameter analysis when combined with other fluorophores

• Typical working dilution: 1:50-200 for most applications

Immunofluorescence Microscopy:

• Enables visualization of DTX3 localization within cells

• Provides high-resolution spatial information

• Typical working dilution: 1:50-200 for cultured cells (ICC) and tissues (IF)

Tissue Cross-Reactivity (TCR) Studies:

• Used to evaluate potential cross-reactivity of therapeutic antibodies

• Important for safety assessment in drug development

Western Blotting:

• Can be used for detection following protein separation

• Usually requires higher antibody concentration (1:300-5000)

When designing experiments, researchers should consider that DTX3 is a regulator of Notch signaling involved in cell-cell communications that regulate cell-fate determinations . FITC-conjugated antibodies are excited by the 488 nm line of an argon laser, with emission collected at 530 nm, making them compatible with standard fluorescence detection systems .

How should I design experiments to study DTX3 expression in different tissue types using FITC-conjugated antibodies?

Designing robust experiments to study DTX3 expression across tissue types requires careful consideration of multiple factors:

Experimental Design Framework:

• Tissue Selection and Processing:

  • Include tissues known to express DTX3 (kidney, testis, brain, ovary)

  • Process tissues consistently (fixation time, temperature, buffer composition)

  • Prepare sections of uniform thickness (5-7 μm recommended)

• Controls Implementation:

  • Positive controls: Mouse kidney/testis tissue (confirmed DTX3 expression)

  • Negative controls: Include secondary-only controls and isotype controls

  • Blocking controls: Pre-incubate antibody with recombinant DTX3 protein

• Staining Protocol Optimization:

  • Perform titration series (1:20 to 1:200 dilutions recommended)

  • Compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Implement nuclear counterstaining (DAPI) for cellular context

• Multiplexed Analysis:

  • Co-stain with cell-type specific markers to identify DTX3-expressing populations

  • Use compatible fluorophores that minimize spectral overlap with FITC

  • Apply spectral unmixing if necessary

When analyzing DTX3 expression across tissues, it's important to note that research has shown DTX3 expression in mouse kidney, ovary, testis, and brain tissues . For human tissues, expression has been documented in kidney and colon tissues . Antibody dilutions should be optimized for each tissue type, with typical IHC dilutions ranging from 1:20 to 1:200 .

What are the considerations for using FITC-conjugated DTX3 antibodies in flow cytometry?

When using FITC-conjugated DTX3 antibodies in flow cytometry, researchers should address several critical considerations to ensure reliable and reproducible results:

Technical Considerations:

• Signal Optimization:

  • Titrate antibody concentration (typically starting at 1:50-1:200)

  • Consider the FITC-labeling index effect on sensitivity and non-specific binding

  • Use appropriate filter sets (excitation ~495 nm, emission ~519 nm)

• Panel Design:

  • Account for FITC spectral overlap with PE and other fluorophores

  • Apply proper compensation when using multiple fluorophores

  • Position FITC in appropriate detection channel (typically FL1)

• Controls and Validation:

  • Include FMO (Fluorescence Minus One) controls

  • Use isotype-matched FITC-conjugated control antibodies

  • Validate with known DTX3-positive cell lines (e.g., hTERT-RPE1, testis-derived cell lines)

• Sample Preparation:

  • Optimize fixation/permeabilization if detecting intracellular DTX3

  • Maintain consistent cell density (1-5 × 10⁶ cells/mL)

  • Minimize autofluorescence through proper washing steps

• Data Analysis:

  • Calculate relative mean fluorescence intensity (MFI) using the equation:
    MFI = (MFI of sample - MFI of isotype control) / MFI of isotype control

  • Establish proper gating strategies based on unstained and single-stained controls

Research has demonstrated that antibody conjugates with higher FITC-labeling indices tend to be more sensitive but are also more likely to yield non-specific binding , which is particularly important to consider when analyzing rare cell populations or cells with low DTX3 expression levels.

How can I optimize FITC-conjugated DTX3 antibody performance in immunofluorescence experiments?

Optimizing FITC-conjugated DTX3 antibody performance in immunofluorescence requires systematic approach to address common challenges:

Optimization Strategy:

• Signal-to-Noise Ratio Improvement:

  • Implement stringent blocking (3-5% BSA or serum from species unrelated to antibody source)

  • Extend washing steps (3-5 washes of 5-10 minutes each)

  • Reduce autofluorescence with sodium borohydride treatment or commercial reducers

• Antibody Concentration Optimization:

  • Perform systematic titration (1:20, 1:50, 1:100, 1:200)

  • Balance signal intensity against background

  • Consider the F/P ratio effect on sensitivity and specificity

• Antigen Retrieval Enhancement:

  • Compare heat-induced epitope retrieval methods:

    • TE buffer pH 9.0 (recommended primary method for DTX3)

    • Citrate buffer pH 6.0 (alternative method)

  • Optimize retrieval time (10-30 minutes)

• Fixation Protocol Refinement:

  • Test multiple fixatives (4% PFA, methanol, acetone)

  • Adjust fixation duration to preserve epitope accessibility

  • Consider mild permeabilization for optimal antibody penetration

• Mounting Media Selection:

  • Use anti-fade mounting media specifically formulated for FITC

  • Avoid mounting media with high pH that can accelerate FITC photobleaching

  • Consider ProLong Gold or similar products that extend fluorescence lifetime

Research indicates that the conjugation of FITC to antibodies can affect binding properties, with higher labeling indices potentially yielding more sensitive detection but increased risk of non-specific staining . For optimal results, select FITC-labeled DTX3 antibodies that balance sensitivity and specificity for your specific application.

What are the common problems with FITC-conjugated antibodies and how can they be resolved?

FITC-conjugated antibodies, including those targeting DTX3, can present several challenges that require specific troubleshooting approaches:

Common Problems and Solutions:

Research has shown that the FITC-labeling index in antibodies is negatively correlated with binding affinity for target antigens . Therefore, when troubleshooting signal issues, consider that antibodies with higher labeling indices may provide increased sensitivity but at the cost of increased non-specific staining. For optimal results, carefully select FITC-labeled DTX3 antibodies with appropriate labeling densities for your specific application.

What are the best storage and handling practices for FITC-conjugated DTX3 antibodies to maintain activity?

Proper storage and handling of FITC-conjugated DTX3 antibodies is critical for maintaining their activity and extending their usable lifespan:

Optimal Storage Conditions:

• Temperature:

  • Store at -20°C for long-term storage (stable for up to one year)

  • Avoid storing at 4°C for extended periods (>1 month)

  • NEVER store FITC conjugates at room temperature

• Buffer Composition:

  • Use PBS with 0.02% sodium azide and 50% glycerol pH 7.3

  • Consider adding 1% BSA as a stabilizer

  • Ensure pH remains stable (7.2-7.4) to prevent FITC degradation

• Aliquoting Strategy:

  • Prepare small single-use aliquots to avoid freeze-thaw cycles

  • Optimal aliquot volume: 10-20 μL

  • Use amber or opaque tubes to protect from light

• Light Exposure Management:

  • Protect from light at all times (wrap tubes in aluminum foil)

  • Minimize exposure during experimental procedures

  • Work under reduced ambient lighting when possible

• Handling During Experiments:

  • Thaw aliquots rapidly at room temperature

  • Keep on ice during experiment setup

  • Return to -20°C immediately after use

  • Never refreeze thawed antibody solutions that have been at room temperature for >2 hours

Research shows that repeated freeze-thaw cycles significantly reduce antibody activity and fluorescence intensity. Studies indicate that FITC-conjugated antibodies typically maintain approximately 85-90% of their original activity after proper storage for one year, but this can decrease to <50% with improper handling . Additionally, exposure to light can cause photobleaching of the FITC molecule, reducing fluorescence intensity by approximately 5-10% per hour of continuous light exposure.

How can FITC-conjugated DTX3 antibodies be used to investigate Notch signaling pathways?

FITC-conjugated DTX3 antibodies offer sophisticated approaches for investigating Notch signaling mechanisms through various advanced applications:

Advanced Research Applications:

• Co-localization Studies:

  • Combine FITC-DTX3 antibodies with antibodies against other Notch pathway components (NOTCH1-4, JAG1/2, DLL1/3/4) using compatible fluorophores

  • Analyze spatial relationships using confocal microscopy with colocalization coefficients (Pearson's, Manders')

  • Investigate DTX3 interactions with ubiquitination machinery components

• Temporal Dynamics Analysis:

  • Design pulse-chase experiments to track DTX3 trafficking following Notch activation

  • Implement live-cell imaging with FITC-conjugated Fab fragments of DTX3 antibodies

  • Correlate DTX3 dynamics with Notch target gene expression (HES1, HEY1)

• Signaling Perturbation Experiments:

  • Measure DTX3 expression changes following γ-secretase inhibitor treatment

  • Assess DTX3 localization shifts in response to Notch ligand stimulation

  • Investigate DTX3 function in Notch-dependent versus Notch-independent contexts

• Quantitative Image Analysis:

  • Apply automated image segmentation to quantify nuclear versus cytoplasmic DTX3 distribution

  • Develop intensity correlation analysis between DTX3 and key Notch effectors

  • Implement machine learning algorithms for pattern recognition in DTX3 distribution

Research indicates that DTX3 functions as a regulator of Notch signaling, which is involved in cell-cell communications that regulate a broad spectrum of cell-fate determinations . By employing FITC-conjugated DTX3 antibodies in these advanced applications, researchers can elucidate the specific mechanisms through which DTX3 influences Notch pathway activity, potentially identifying novel therapeutic targets for Notch-dependent disorders.

What approaches can be used to validate the specificity of FITC-conjugated DTX3 antibodies in complex experimental systems?

Validating the specificity of FITC-conjugated DTX3 antibodies in complex experimental systems requires a multi-faceted approach combining molecular, cellular, and analytical techniques:

Comprehensive Validation Framework:

• Genetic Validation Approaches:

  • CRISPR/Cas9 knockout of DTX3 in relevant cell lines

  • siRNA/shRNA knockdown with quantitative assessment of signal reduction

  • Overexpression of tagged DTX3 constructs to confirm co-localization with antibody signal

• Biochemical Validation Methods:

  • Pre-absorption controls with recombinant DTX3 protein

  • Peptide competition assays using immunogenic peptide (e.g., aa201-301 region)

  • Western blot correlation with immunofluorescence patterns

• Cross-Platform Validation:

  • Compare DTX3 detection across multiple techniques (IF, flow cytometry, WB)

  • Validate using multiple antibodies targeting different DTX3 epitopes

  • Correlate protein detection with mRNA expression (RT-qPCR or RNA-seq)

• Analytical Validation:

  • Implement quantitative colocalization analysis with known DTX3 binding partners

  • Perform spectral analysis to distinguish true FITC signal from autofluorescence

  • Apply signal-to-noise ratio measurements across different experimental conditions

• Species Cross-Reactivity Assessment:

  • Test antibody performance across multiple species (human, mouse, rat if applicable)

  • Compare staining patterns with evolutionary conservation of DTX3 sequence

  • Validate in tissues with known differential expression patterns

Research indicates that DTX3 is expressed in specific tissues including kidney, testis, ovary, and brain . Validation experiments should include these tissues as positive controls. Additionally, when validating in immunohistochemistry applications, it's recommended to compare TE buffer pH 9.0 with citrate buffer pH 6.0 for antigen retrieval to ensure optimal epitope accessibility .

How can multiplexed imaging with FITC-conjugated DTX3 antibodies be optimized for studying protein interaction networks?

Optimizing multiplexed imaging with FITC-conjugated DTX3 antibodies for protein interaction network analysis requires sophisticated strategies to maximize information yield while minimizing technical artifacts:

Advanced Multiplexing Strategies:

• Spectral Selection and Compensation:

  • Carefully select fluorophores to minimize spectral overlap with FITC (Ex/Em: 495/519 nm)

  • Recommended compatible fluorophores: Cy5 (649/670 nm), Texas Red (596/615 nm), DAPI (358/461 nm)

  • Implement linear unmixing algorithms to separate overlapping signals

  • Prepare single-stained controls for accurate spectral fingerprinting

• Sequential Multiplexing Techniques:

  • Apply iterative staining-imaging-bleaching cycles:

    • Image FITC-DTX3 and compatible fluorophores

    • Chemically bleach fluorophores

    • Restain with new antibody panel

    • Register and overlay images computationally

  • Consider tyramide signal amplification for weak signals

• Advanced Microscopy Methods:

  • Implement super-resolution techniques (STED, STORM, SIM) for nanoscale interaction analysis

  • Apply FRET (Förster Resonance Energy Transfer) to detect direct DTX3 protein interactions

  • Utilize light-sheet microscopy for 3D visualization of interaction networks in intact tissues

• Quantitative Analysis Frameworks:

  • Develop computational pipelines for colocalization analysis:

    • Pearson's correlation coefficient

    • Manders' overlap coefficient

    • Object-based colocalization analysis

  • Implement proximity ligation assays to confirm direct DTX3 interactions

  • Apply graph theory to map DTX3-centric protein interaction networks

• Validation Controls for Multiplexed Systems:

  • Include multi-color beads for chromatic aberration correction

  • Employ fluorescent protein fusion constructs as positive interaction controls

  • Use structurally unrelated protein pairs as negative interaction controls

Research has shown that DTX3 functions as a regulator of Notch signaling, which involves complex interaction networks with multiple proteins . When designing multiplexed imaging experiments, it's important to consider that the FITC-labeling index in antibodies can affect both sensitivity and specificity . Antibodies with optimal F/P ratios (3-6 FITC molecules per antibody) typically provide the best balance between signal intensity and specific binding for multiplexed applications .

How can FITC-conjugated DTX3 antibodies be applied to investigate tissue-specific expression patterns in disease models?

Applying FITC-conjugated DTX3 antibodies to investigate tissue-specific expression patterns in disease models requires comprehensive experimental design strategies:

Experimental Design Framework:

• Disease Model Selection and Validation:

  • Choose appropriate models related to Notch signaling dysregulation

  • Consider genetic models (transgenic, knockout) and induced models (chemical, surgical)

  • Validate model pathology against human disease characteristics

• Tissue Panel and Processing Strategy:

  • Include comprehensive tissue panel covering:

    • Known DTX3-expressing tissues (kidney, testis, ovary, brain)

    • Disease-relevant tissues based on pathophysiology

    • Control tissues with minimal DTX3 expression

  • Standardize tissue collection and fixation protocols to minimize variability

• Multiplex Staining Design:

  • Combine FITC-DTX3 antibody with:

    • Cell-type specific markers to identify expressing populations

    • Disease-specific markers to correlate with pathology

    • Signaling pathway components to establish functional context

  • Implement sequential staining for incompatible antibody combinations

• Quantitative Analysis Methodology:

  • Develop tissue cytometry approaches:

    • Automated whole-slide scanning

    • Machine learning-based cell classification

    • Region-specific quantification of DTX3 expression

  • Correlate DTX3 expression with histopathological features

• Complementary Validation Methods:

  • Confirm tissue expression patterns with orthogonal techniques:

    • Laser capture microdissection with RT-qPCR

    • Single-cell RNA sequencing

    • Western blot analysis of tissue lysates

Research indicates that DTX3 antibodies have been successfully used in immunohistochemistry applications with optimal dilutions ranging from 1:20-1:200 . When investigating disease models, it's important to note that antibodies with higher FITC-labeling indices tend to be more sensitive but are also more likely to yield non-specific staining , which can be particularly problematic in tissues with inflammation or increased autofluorescence.

What are the considerations for using FITC-conjugated DTX3 antibodies in conjunction with other antibody-drug conjugates for mechanistic studies?

Combining FITC-conjugated DTX3 antibodies with antibody-drug conjugates (ADCs) for mechanistic studies requires careful consideration of multiple technical and biological factors:

Key Considerations:

• Compatibility Assessment:

  • Evaluate potential interactions between FITC-conjugated antibodies and ADC components

  • Consider steric hindrance effects when targeting closely located epitopes

  • Assess whether FITC fluorescence may be quenched by proximity to ADC payloads

• Sequential Application Strategy:

  • Design protocols for sequential rather than simultaneous application when necessary

  • Determine optimal sequence (FITC-antibody followed by ADC or vice versa)

  • Establish appropriate washing steps to remove unbound antibodies

• Advanced Microscopy Methods:

  • Implement super-resolution techniques to visualize nanoscale distributions

  • Utilize live-cell imaging to monitor temporal dynamics of internalization

  • Apply FRET or BRET techniques to assess proximity between DTX3 and ADC targets

• Controls for Mechanistic Studies:

  • Include non-targeting FITC-conjugated antibodies to assess non-specific effects

  • Use unconjugated primary antibodies alongside conjugated versions to evaluate conjugation effects

  • Implement antibody fragments (Fab) as size-matched controls

• Analytical Framework for Mechanistic Insights:

  • Develop quantitative colocalization analysis with spatial statistics

  • Apply pharmacokinetic modeling to understand ADC-target interactions

  • Correlate imaging data with functional outcomes (cell viability, signaling pathway activity)

Research in the field of ADCs has demonstrated that the conjugation process can affect antibody binding properties . When designing studies combining FITC-conjugated DTX3 antibodies with ADCs, it's important to consider that both conjugates may exhibit altered binding kinetics compared to their unconjugated counterparts . Studies have shown that ADCs can be effectively used to investigate non-oncological applications, including inflammatory conditions and immunological disorders , which may overlap with DTX3-related Notch signaling mechanisms.

What advanced data analysis approaches are recommended for quantifying DTX3 expression using FITC-conjugated antibodies?

Advanced data analysis for quantifying DTX3 expression using FITC-conjugated antibodies requires sophisticated computational approaches to extract meaningful biological insights:

Advanced Analytical Framework:

• Multidimensional Image Analysis:

  • Implement 3D/4D image analysis for volumetric assessment of DTX3 expression

  • Apply deconvolution algorithms to improve signal resolution

  • Utilize machine learning-based segmentation for accurate cell/organelle identification:

    • Convolutional neural networks for feature detection

    • Random forest classifiers for pixel/voxel classification

    • Active contour models for boundary detection

• Quantitative Expression Metrics:

  • Calculate multiscale metrics beyond simple intensity measurements:

    • Integrated density (product of area and mean gray value)

    • Nuclear/cytoplasmic ratio of DTX3 expression

    • Correlation with cell morphological features

  • Implement relative expression calculations:

    • Normalized to housekeeping proteins

    • Z-score normalization across experimental conditions

    • Comparison to calibrated standards

• Spatial Statistics and Pattern Analysis:

  • Apply spatial statistics to characterize DTX3 distribution patterns:

    • Ripley's K-function for clustering analysis

    • Nearest neighbor distance analysis

    • Quadrat analysis for regional distribution

  • Develop topological data analysis to identify structural patterns

• Multi-parametric Correlation Analysis:

  • Integrate DTX3 expression data with:

    • Transcriptomic profiles from matched samples

    • Clinical/pathological parameters in disease models

    • Other Notch pathway component expression levels

  • Apply dimensionality reduction techniques (PCA, t-SNE, UMAP) for pattern recognition

• Reproducibility and Standardization:

  • Implement batch correction algorithms to account for experimental variability

  • Develop standardized reporting following MISFISHIE guidelines

  • Utilize fluorescence calibration standards (beads, slides) for absolute quantification

Research indicates that optimization of image acquisition parameters is critical for accurate quantification when using FITC-conjugated antibodies. The literature shows that photobleaching can significantly impact quantification accuracy, with FITC showing approximately 5-10% signal reduction per minute under continuous illumination . Additionally, when analyzing DTX3 expression in relation to the Notch signaling pathway, it's important to consider its regulatory role in cell-cell communications that influence cell-fate determinations .