DTX1 Antibody, FITC conjugated

What is DTX1 and what are its primary cellular functions?

DTX1 (Deltex1) functions as an E3 ubiquitin ligase protein in vivo, mediating ubiquitination and promoting degradation of MEKK1. It serves as a crucial regulator of the Notch signaling pathway, which is involved in cell-cell communications and regulates a broad spectrum of cell-fate determinations . Interestingly, DTX1 can act as both a positive and negative regulator of Notch, depending on the developmental and cellular context. The protein is involved in several biological processes including neurogenesis, lymphogenesis, and myogenesis. Additionally, it may play a role in marginal zone B (MZB) cell differentiation and promotes B-cell development while suppressing T-cell development, suggesting it can antagonize NOTCH1 .

What is the significance of FITC conjugation in DTX1 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides a fluorescent tag that enables direct visualization of the DTX1 protein in various applications without requiring secondary antibodies. The FITC fluorophore emits green fluorescence when excited, allowing detection through fluorescence microscopy, flow cytometry, and other fluorescence-based techniques . This direct conjugation simplifies experimental protocols by eliminating the need for secondary antibody incubation steps, reducing background noise, and minimizing cross-reactivity issues. FITC-conjugated antibodies are particularly valuable for multicolor immunostaining experiments where multiple targets need to be visualized simultaneously using different fluorophores .

What are the typical applications for DTX1 FITC-conjugated antibodies in research?

DTX1 FITC-conjugated antibodies can be utilized in multiple research applications, including:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) to visualize DTX1 expression patterns in tissue sections

• Flow cytometry for intracellular staining to quantify DTX1 expression in cell populations

• Immunocytochemistry to localize DTX1 within cultured cells

• CyTOF (mass cytometry) for high-dimensional analysis of DTX1 along with other cellular markers

• Western blot analysis for detecting DTX1 protein levels and molecular weight validation

The recommended dilution for immunofluorescence applications typically ranges from 1:50-1:200, though this may vary depending on the specific antibody formulation and experimental conditions .

How should I design experiments to study DTX1's role in Notch signaling using FITC-conjugated antibodies?

When designing experiments to investigate DTX1's role in Notch signaling using FITC-conjugated antibodies, consider implementing the following methodological approach:

First, establish cellular models with varying levels of DTX1 expression (overexpression, knockdown, or knockout) to observe consequent effects on Notch pathway components. Based on research findings, expressions of Notch1, Jagged1, and HES1 are significantly down-regulated in cells overexpressing DTX1, while these molecules increase in DTX1-knockdown cells (p<0.01) . To validate the functional relationship between DTX1 and Notch signaling, incorporate gamma-secretase inhibitor (GSI) treatment as a Notch pathway blocker. Research has shown that GSI treatment (compound E, 10 μM for 3 days) reduces cell proliferation to 62% compared to untreated controls .

For visualization and quantification, use the FITC-conjugated DTX1 antibody in:

• Immunofluorescence to co-localize DTX1 with Notch pathway components

• Flow cytometry to quantify changes in DTX1 levels following Notch activation/inhibition

• Western blot analysis with parallel samples to correlate protein levels

Include appropriate controls: isotype controls, secondary antibody-only controls, and positive controls with known DTX1 expression patterns to ensure specificity and reliability of your FITC-conjugated antibody staining.

What protocols should be followed for optimal DTX1 detection in flow cytometry using FITC-conjugated antibodies?

For optimal detection of DTX1 in flow cytometry using FITC-conjugated antibodies, follow this methodological protocol:

• Cell preparation: Harvest 1×10^6 cells per sample and wash twice with PBS containing 1% BSA.

• Fixation and permeabilization: Since DTX1 is primarily an intracellular protein, fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.1% Triton X-100 or a commercial permeabilization buffer for 10 minutes .

• Blocking: Incubate cells with 5% normal serum (matched to the species in which the secondary antibody was raised) for 30 minutes to reduce non-specific binding.

• Primary antibody staining: Incubate cells with the FITC-conjugated DTX1 antibody at the optimal concentration (typically starting with manufacturer's recommendation, e.g., 5-10 μg/ml) for 30-60 minutes at room temperature in the dark .

• Washing: Wash cells 3 times with PBS containing 1% BSA to remove unbound antibody.

• Controls: Include appropriate controls:

  • Unstained cells to establish autofluorescence

  • Isotype control-FITC to determine non-specific binding

  • Positive control (cells known to express DTX1)

  • Negative control (DTX1-negative cells or DTX1-knockdown cells)

• Instrument setup: Configure flow cytometer with appropriate filters for FITC detection (excitation ~490 nm, emission ~520 nm).

• Analysis: Gate on viable cells first, then analyze FITC signal intensity. For DTX1 expression studies in breast cancer or T-regulatory cells, correlate with other relevant markers such as CD4, CD25, and Foxp3 .

What are the critical steps for successful immunohistochemistry with DTX1 FITC-conjugated antibodies?

Successful immunohistochemistry (IHC) with DTX1 FITC-conjugated antibodies requires attention to several critical steps:

• Tissue preparation and antigen retrieval:

  • Fix tissues appropriately (10% neutral buffered formalin is standard)

  • For paraffin-embedded sections, perform deparaffinization and rehydration

  • Conduct heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • This step is crucial as improper antigen retrieval can lead to false negative results, particularly for intracellular targets like DTX1

• Blocking steps:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block endogenous biotin if using biotin-based detection systems

  • Use 5-10% normal serum (from the same species as the secondary antibody) to reduce background staining

  • For FITC-conjugated antibodies specifically, include an avidin/biotin blocking step if needed

• Antibody dilution and incubation:

  • Determine optimal antibody dilution through titration (recommended starting dilution 1:50-1:200)

  • Incubate sections with diluted antibody for appropriate time (typically overnight at 4°C or 1-2 hours at room temperature)

  • Maintain dark conditions to prevent photobleaching of the FITC fluorophore

• Washing and counterstaining:

  • Wash thoroughly between steps with TBS or PBS to remove unbound antibody

  • Use DAPI or another blue nuclear counterstain that won't interfere with the green FITC signal

  • Mount with anti-fade mounting medium specifically designed for fluorescence preservation

• Controls and validation:

  • Include positive control tissues (human breast cancer tissues with known DTX1 expression)

  • Include negative controls (omitting primary antibody)

  • Consider using tissues from DTX1-knockout models as specificity controls

How can I distinguish between specific and non-specific binding when using DTX1 FITC-conjugated antibodies?

Distinguishing between specific and non-specific binding when using DTX1 FITC-conjugated antibodies requires implementation of several validation strategies:

• Comprehensive controls:

  • Use isotype-matched FITC-conjugated control antibodies to identify non-specific binding patterns

  • Include blocking peptide competition assays where the antibody is pre-incubated with recombinant DTX1 protein (such as E. coli-derived recombinant human DTX1, Met1-Phr147, Accession #Q86Y01) - specific signals should be significantly reduced

  • Implement genetic controls using DTX1 knockout or knockdown models alongside wild-type samples

• Signal pattern analysis:

  • Specific DTX1 binding should follow established subcellular localization patterns

  • Compare staining patterns with published literature on DTX1 localization

  • Non-specific binding often presents as diffuse background or unexpected subcellular localization

• Cross-validation with multiple detection methods:

  • Confirm findings using multiple antibody clones targeting different DTX1 epitopes

  • Validate protein expression using complementary techniques (e.g., RT-PCR for mRNA expression)

  • Western blot analysis should show bands of the expected molecular weight (~60 kDa for DTX1)

• Optimization protocols:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Test multiple blocking reagents (5-10% normal serum, commercial blocking buffers, protein-free blockers)

  • Adjust incubation times and temperatures to enhance specific binding while minimizing non-specific interactions

• Analysis of signal distribution across sample populations:

  • Specific binding should demonstrate biologically reasonable variation across samples

  • Correlate DTX1 expression with known biological contexts (e.g., lower in breast cancer tissues compared to fibroadenoma tissues)

What are the common sources of false-positive and false-negative results when using FITC-conjugated DTX1 antibodies?

When working with FITC-conjugated DTX1 antibodies, researchers should be aware of several potential sources of false results:

Sources of false-positive results:

• Autofluorescence issues:

  • Naturally fluorescent compounds in tissues (e.g., lipofuscin, elastin) may emit in the same spectrum as FITC

  • Formalin fixation can create fluorescent artifacts in tissues

  • Solution: Include unstained controls and consider autofluorescence quenching reagents

• Cross-reactivity:

  • Antibodies raised against DTX1 may recognize related proteins like DTX4 due to sequence homology

  • Solution: Validate antibody specificity with recombinant proteins and genetic controls

• Non-specific binding mechanisms:

  • Hydrophobic interactions between antibody and sample components

  • Fc receptor binding in immune cell-rich tissues

  • Solution: Use appropriate blocking reagents and Fc receptor blockers when needed

• Technical artifacts:

  • Over-fixation can create fluorescent precipitates

  • Inadequate washing can leave residual unbound antibody

  • Solution: Optimize fixation protocols and implement rigorous washing steps

Sources of false-negative results:

• Epitope masking:

  • Inadequate antigen retrieval, particularly for formalin-fixed tissues

  • Protein-protein interactions blocking antibody access to DTX1

  • Solution: Optimize antigen retrieval methods (test both heat-induced and enzymatic methods)

• Antibody limitations:

  • Epitope not exposed in native conformation of the protein

  • Epitope destroyed during sample processing

  • Solution: Try antibodies targeting different DTX1 epitopes

• Technical issues:

  • FITC photobleaching due to prolonged light exposure

  • Using improper filters for detection

  • Solution: Minimize light exposure, use anti-fade mounting media, and verify microscope filter settings

• Biological factors:

  • Low expression levels of DTX1 in certain contexts (e.g., advanced breast cancer)

  • Post-translational modifications altering epitope recognition

  • Solution: Increase antibody concentration, use signal amplification techniques, or employ more sensitive detection systems

How should storage and handling conditions be optimized for maintaining FITC-conjugated DTX1 antibody effectiveness?

To maintain optimal effectiveness of FITC-conjugated DTX1 antibodies, implement these evidence-based storage and handling practices:

• Temperature conditions:

  • Store antibodies at recommended temperatures, typically 4°C for short-term storage and -20°C or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles; aliquot antibodies upon receipt

  • Research demonstrates that each freeze-thaw cycle can reduce antibody activity by 5-10%

• Light protection:

  • FITC is particularly susceptible to photobleaching; store in amber vials or wrap containers in aluminum foil

  • Minimize exposure to light during all handling steps

  • Work under reduced ambient lighting when preparing samples for microscopy or flow cytometry

• Buffer composition:

  • Verify that the antibody is stored in an appropriate buffer (typically PBS with stabilizers)

  • Some formulations may include sodium azide as a preservative; note that this can interfere with certain applications

  • Consider adding stabilizing proteins (BSA 1-5%) if not already present in commercial formulations

• Contamination prevention:

  • Use sterile technique when handling antibody solutions

  • Filter buffers used for dilution to remove particulates

  • Never return unused antibody to the original container

• Record-keeping:

  • Document date of receipt, aliquoting, and each use

  • Track lot numbers and correlate with experimental results

  • Document number of freeze-thaw cycles if applicable

• Stability assessment:

  • Periodically test antibody performance on positive control samples

  • Consider including a reference standard curve in flow cytometry applications

  • If significant loss of signal is observed, obtain fresh antibody

A properly maintained FITC-conjugated DTX1 antibody should maintain activity for at least 12 months when stored according to manufacturer recommendations. Degradation may be detected as decreased fluorescence intensity, increased background, or loss of specific binding pattern.

How does DTX1 expression correlate with breast cancer progression, and how can FITC-conjugated antibodies help track these changes?

DTX1 expression demonstrates a significant inverse correlation with breast cancer progression, offering potential as a biomarker for disease advancement. Research has revealed that DTX1 levels in breast cancer tissues are markedly lower compared to fibroadenoma tissues and peri-neoplastic breast tissues (p<0.01) . This decreased expression pattern correlates with several clinical parameters of disease severity:

FITC-conjugated DTX1 antibodies provide powerful tools for tracking these expression changes through multiple methodological approaches:

• Flow cytometric analysis:

  • Enables quantitative measurement of DTX1 protein levels at the single-cell level

  • Allows correlation with other prognostic markers in the same sample

  • Permits identification of DTX1-low cell subpopulations within heterogeneous tumors

• Immunohistochemical assessment:

  • Visualization of DTX1 expression patterns within the tumor microenvironment

  • Correlation with spatial information (e.g., tumor margins vs. core)

  • Potential for automated image analysis to quantify expression levels across large cohorts

• Functional studies:

  • Lower levels of DTX1 have been shown to promote breast cancer cell proliferation, migration, and invasion in vitro

  • FITC-conjugated antibodies can track DTX1 levels after experimental manipulations

  • Enables correlation between DTX1 expression and cellular behavior in real-time analyses

Notably, research has identified DTX1 as a potential independent prognostic marker, with lower expression recognized as an impact factor for metastasis-free survival in breast cancer patients . This suggests that monitoring DTX1 expression using FITC-conjugated antibodies could provide valuable information for predicting disease progression and treatment response.

What is the role of DTX1 in T-regulatory cell stability, and how can FITC-conjugated antibodies be used to investigate this function?

DTX1 plays a critical role in maintaining regulatory T cell (Treg) stability through a mechanism involving the antagonism of HIF-1α and subsequent preservation of Foxp3 expression. This regulatory relationship has significant implications for immune tolerance and autoimmunity.

The mechanism of DTX1's function in Treg stability involves:

• HIF-1α regulation: DTX1 promotes the degradation of HIF-1α, a transcription factor that can negatively impact Foxp3 stability .

• Foxp3 maintenance: By antagonizing HIF-1α, DTX1 sustains the expression of Foxp3 protein in Tregs in vivo, which is essential for maintaining their suppressive function .

• Functional consequences: While DTX1-deficient Tregs (Dtx1-/- Tregs) remain effective at inhibiting CD4+CD25- T-cell activation in vitro, their suppressive ability is significantly impaired in vivo . This discrepancy highlights the context-dependent nature of DTX1's role.

FITC-conjugated DTX1 antibodies offer several methodological approaches to investigate this function:

• Co-localization studies:

  • Use multicolor flow cytometry to simultaneously detect FITC-conjugated DTX1 antibodies alongside markers for Tregs (CD4, CD25, Foxp3)

  • Perform confocal microscopy to visualize the subcellular localization of DTX1 in relation to HIF-1α and Foxp3

• Quantitative analysis:

  • Monitor changes in DTX1 expression levels in Tregs under various conditions (hypoxia, inflammation, etc.)

  • Correlate DTX1 expression with Foxp3 stability and suppressive function

• Time-course experiments:

  • Track DTX1 and Foxp3 expression in adoptively transferred Tregs using FITC-conjugated antibodies

  • Experimental evidence shows that Foxp3 expression diminishes when Dtx1-/- Tregs are co-transferred with effector T cells in vivo

• Genetic validation:

  • Use FITC-conjugated DTX1 antibodies to confirm knockdown or knockout efficiency in experimental models

  • Research has demonstrated that knockout of HIF-1α restores Foxp3 stability and rescues the defective suppressive activity in Dtx1-/- Treg cells in vivo

This research area has significant implications for understanding autoimmune diseases and developing Treg-based therapies, where maintaining Treg stability is essential for therapeutic efficacy.

How does DTX1 interact with the Notch signaling pathway, and what methodologies using FITC-conjugated antibodies can elucidate these mechanisms?

DTX1 exhibits a complex, context-dependent relationship with the Notch signaling pathway, functioning as both a positive and negative regulator. FITC-conjugated DTX1 antibodies enable sophisticated methodological approaches to investigate these interactions.

Dual regulatory mechanisms of DTX1 in Notch signaling:

• Positive regulation:

  • DTX1 can enhance Notch signaling in certain developmental contexts

  • Functions as a ubiquitin ligase that may target specific negative regulators of Notch

• Negative regulation:

  • DTX1 can inhibit Notch signaling in other contexts, particularly in lymphocyte development

  • Promotes B-cell development at the expense of T-cell development, suggesting antagonism of NOTCH1

  • Mediates the antineural activity of Notch by potentially inhibiting transcriptional activation mediated by MATCH1

• Molecular targets:

  • Research indicates that DTX1 mediates ubiquitination and promotes degradation of MEKK1

  • Expression of DTX1 leads to significant down-regulation of Notch pathway components including Notch1, Jagged1, and HES1

Methodological approaches using FITC-conjugated antibodies:

• Protein interaction studies:

  • Co-immunoprecipitation followed by detection with FITC-conjugated DTX1 antibodies

  • Proximity ligation assays to visualize interactions between DTX1 and Notch pathway components

  • FRET analysis to detect direct interactions in live cells

• Expression correlation analysis:

  • Multicolor flow cytometry using FITC-conjugated DTX1 antibodies alongside antibodies against Notch pathway components

  • This approach can reveal inverse correlations, such as the observed down-regulation of Notch1, Jagged1, and HES1 in cells overexpressing DTX1

• Functional validation:

  • Pharmacological manipulation using gamma-secretase inhibitors (GSI, compound E, 10 μM) to establish a Notch-off state

  • Measuring the impact on cell proliferation and migration while tracking DTX1 expression

  • Research shows GSI treatment reduces proliferation to 62% of control cells and significantly inhibits wound closure in migration assays (reduced by 15% at 24h and 30% at 48h)

• Dynamic expression tracking:

  • Time-lapse microscopy with FITC-conjugated DTX1 antibodies to monitor expression changes during Notch activation/inhibition

  • Single-cell analysis to capture heterogeneity in DTX1-Notch pathway interactions

Understanding these complex interactions has significant implications for developmental biology, cancer research, and immunology, as the DTX1-Notch axis influences cell fate decisions across multiple tissues and disease states.

How should researchers quantify and analyze DTX1 expression levels using FITC-conjugated antibodies in different experimental systems?

Accurate quantification and analysis of DTX1 expression using FITC-conjugated antibodies requires system-specific methodologies and appropriate analytical approaches:

For Flow Cytometry Analysis:

For Immunofluorescence/Immunohistochemistry Analysis:

• Image acquisition parameters:

  • Standardize exposure times, gain settings, and detector sensitivity

  • Capture multiple fields per sample (minimum 5-10 fields)

  • Use the same magnification across comparable samples

• Quantification approaches:

  • Mean fluorescence intensity measurement within regions of interest

  • Nuclear/cytoplasmic ratio of DTX1 expression

  • Colocalization coefficients when examining interaction with Notch pathway components

• Image analysis software:

  • ImageJ/FIJI with appropriate plugins for fluorescence quantification

  • CellProfiler for automated cell segmentation and intensity measurement

  • QuPath for advanced tissue analysis, particularly in breast cancer studies

For Western Blot Analysis:

• Normalization strategy:

  • Always normalize DTX1 signal to loading controls (β-actin, GAPDH)

  • Consider using total protein normalization for more accurate quantification

  • Include recombinant DTX1 standards when absolute quantification is needed

• Densitometric analysis:

  • Use linear range of detection for quantification

  • Subtract background from all measurements

  • Present data as fold-change relative to control conditions

Statistical Analysis Across Platforms:

• Appropriate statistical tests:

  • For comparing DTX1 expression between groups (e.g., breast cancer vs. normal tissue), use t-tests or ANOVA with appropriate post-hoc tests

  • For correlation with clinical parameters, use chi-square tests or Fisher's exact test

  • For survival analysis related to DTX1 expression, employ Kaplan-Meier curves with log-rank tests

• Presentation standards:

  • Include both representative images and quantitative graphs

  • Provide clear indication of sample size and biological replicates

  • Always indicate statistical significance (p-values) and specify tests used

What are the critical considerations when interpreting DTX1 expression data in the context of Notch signaling pathway alterations?

• Context-dependent regulatory relationships:

  • DTX1 can function as either a positive or negative regulator of Notch signaling depending on cellular context

  • Always consider the specific cell type, developmental stage, and disease state when interpreting results

  • In breast cancer cells, DTX1 overexpression correlates with downregulation of Notch1, Jagged1, and HES1, suggesting a negative regulatory role in this context

• Pathway component analysis:

  • Examine multiple Notch pathway components simultaneously (Notch1-4, ligands, downstream targets)

  • Consider using a pathway activity score rather than individual protein levels

  • Canonical Notch targets like HES1 provide functional readouts of pathway activity

• Temporal dynamics considerations:

  • Notch signaling operates with temporal oscillations in many contexts

  • Single time-point measurements may miss important dynamic relationships

  • Consider time-course experiments when resources permit

• Post-translational modification analysis:

  • DTX1 functions as an E3 ubiquitin ligase affecting protein stability

  • Changes in total protein levels may not reflect changes in activity

  • Consider examining ubiquitination status of relevant targets (e.g., MEKK1)

• Pathway modulation validation:

  • Pharmacological inhibitors (e.g., γ-secretase inhibitors/GSIs) provide powerful tools for validation

  • Research shows GSI treatment (compound E, 10 μM) reduces cell proliferation to 62% of control levels

  • Genetic approaches (siRNA, CRISPR) targeting DTX1 or Notch components offer complementary validation

• Statistical approach for contradictory data:

  • When findings appear contradictory, stratify analyses by relevant variables

  • Consider employing multivariate analyses to identify confounding factors

  • Test for interaction effects between DTX1 and other pathway components

• Biological outcome correlation:

  • Correlate pathway alterations with functional outcomes (proliferation, migration, differentiation)

  • In breast cancer studies, lower DTX1 expression correlates with advanced tumor grade (p=0.017), advanced clinical stage (p=0.031), positive lymph node metastasis (p=0.009), and worse prognosis

  • This correlation provides context for interpreting molecular changes

How can researchers integrate DTX1 expression data from multiple experimental platforms to develop comprehensive biological insights?

Integrating DTX1 expression data across multiple experimental platforms requires systematic methodological approaches to synthesize cohesive biological insights:

• Cross-platform normalization strategies:

  • Develop relative expression scales that can be compared across platforms

  • Use common reference samples across all platforms

  • Consider computational normalization methods (z-scores, quantile normalization)

  • When analyzing publicly available datasets, use established batch correction algorithms

• Multi-omics integration framework:

  • Correlate protein-level DTX1 data (from FITC-antibody studies) with transcriptomic data

  • Integrate with epigenetic data to understand regulatory mechanisms

  • Connect with interactome data to place DTX1 in protein-protein interaction networks

  • Link to functional genomics screens investigating Notch pathway or ubiquitination

• Pathway modeling approaches:

  • Develop quantitative models incorporating DTX1's dual role in Notch signaling

  • Use experimental data to parameterize ordinary differential equation (ODE) models

  • Employ Boolean network models for qualitative understanding of regulatory relationships

  • Test model predictions with targeted experiments

• Correlation with clinical parameters:

  • Integrate experimental findings with patient data when available

  • Research demonstrates that lower DTX1 expression correlates with advanced tumor grade, clinical stage, positive lymph node metastasis, and high Ki-67 index in breast cancer

  • Calculate multivariate models including DTX1 alongside established biomarkers

• Visualization and analysis tools:

  • Use pathway visualization tools (Cytoscape, PathVisio) to map experimental findings

  • Employ dimensionality reduction techniques to identify patterns across datasets

  • Develop integrated heatmaps showing DTX1 and related gene/protein expression

  • Consider machine learning approaches for pattern recognition across complex datasets

• Validation across experimental systems:

  • Confirm key findings in multiple cell lines or model organisms

  • Validate in primary cells or tissues when possible

  • For breast cancer studies, compare findings between cell lines and patient samples

  • For immune cell studies, compare findings between in vitro and in vivo systems

• Comprehensive hypothesis development:

  • Synthesize findings into testable hypotheses about DTX1 function

  • For example, integration of breast cancer data suggests DTX1 as a tumor suppressor acting via Notch pathway inhibition

  • For T-regulatory cells, integrated data suggests DTX1 maintains Foxp3 stability by antagonizing HIF-1α

  • Design validation experiments targeting specific nodes in the proposed mechanisms

By implementing these integration strategies, researchers can develop a systems-level understanding of DTX1 biology that transcends the limitations of any single experimental approach or platform.