DTX4 Antibody, FITC conjugated

What is DTX4 and what are its known biological functions?

DTX4, also known as E3 ubiquitin-protein ligase DTX4, Protein deltex-4, Deltex4, or RING finger protein 155 (RNF155), functions as a critical E3 ubiquitin ligase in cellular signaling pathways. The protein has been identified with UniProt ID Q9Y2E6 and plays a significant role in the regulation of type I interferon signaling .

DTX4's most well-characterized function involves its recruitment by the pattern-recognition receptor NLRP4 to target the TANK-binding kinase 1 (TBK1) for Lys48 (K48)-linked polyubiquitination, specifically at Lys670. This post-translational modification leads to the degradation of TBK1, thereby inhibiting type I interferon signaling and providing a negative regulatory mechanism for antiviral immunity . This NLRP4-DTX4 axis represents an important signaling cascade for maintaining immune homeostasis during antiviral innate immunity responses.

How does the FITC conjugation affect antibody applications and detection methods?

FITC (Fluorescein isothiocyanate) conjugation provides several advantages for DTX4 antibody applications in fluorescence-based detection systems. The FITC fluorophore has excitation/emission wavelengths of 499/515 nm and is compatible with the 488 nm laser line commonly found in flow cytometers and fluorescence microscopes .

The conjugation process chemically links FITC directly to the antibody molecule, eliminating the need for secondary antibody detection steps in experimental protocols. This direct labeling approach offers several methodological advantages:

• Reduced protocol complexity and time requirements

• Elimination of potential cross-reactivity issues associated with secondary antibodies

• Compatibility with multicolor detection systems where multiple targets can be visualized simultaneously

• The conjugation process may potentially mask or alter critical epitope binding regions

• FITC is relatively susceptible to photobleaching compared to other fluorophores

• The fluorophore exhibits pH sensitivity that may affect signal intensity in different buffer conditions

For optimal results, researchers should determine appropriate concentration through titration experiments and incorporate appropriate controls to account for these considerations .

What are the optimal storage conditions for maintaining DTX4 antibody functionality?

Proper storage is critical for maintaining the functionality of FITC-conjugated DTX4 antibodies. The following recommendations should be followed to preserve antibody activity and fluorescence properties:

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Aliquot the antibody solution to avoid repeated freeze-thaw cycles that can degrade protein structure and reduce antigen-binding capacity

• When storing working dilutions, maintain at 4°C and use within 1-2 weeks

• Protect from prolonged light exposure during handling and experimental procedures

• Store in recommended buffer conditions (typically PBS with glycerol and preservatives)

The manufacturer specifications indicate that the antibody is supplied in liquid form with a buffer composition of 0.01M PBS (pH 7.4), 0.03% Proclin 300, and 50% Glycerol. This formulation provides stability during freezing while preventing microbial contamination .

What experimental techniques can effectively utilize DTX4 antibody, FITC conjugated?

FITC-conjugated DTX4 antibodies can be employed in multiple research applications, with each requiring specific methodological considerations:

Flow Cytometry:

• Optimal for analyzing DTX4 expression in heterogeneous cell populations

• Typical working dilution range: 1:50-1:200 (should be determined empirically)

• Include appropriate isotype control (FITC-conjugated rabbit IgG)

• Consider compensation when using multiple fluorophores

• Fixation and permeabilization required for intracellular staining

Immunofluorescence Microscopy:

• Enables visualization of DTX4 localization within cells

• Fixed cells require permeabilization to access intracellular DTX4

• Standard dilution range: 1:100-1:500

• Include DAPI nuclear counterstain for structural reference

• Mount with anti-fade medium to reduce photobleaching

ELISA:

• The commercial product is reported to be applicable for ELISA techniques

• Recommended optimization for plate coating, blocking, and detection parameters

• Typical concentration range: 1-10 μg/ml for coating

The polyclonal nature of the available antibody provides robust recognition but may require more extensive validation to ensure specificity compared to monoclonal alternatives.

How can researchers validate DTX4 antibody specificity in their experimental systems?

Validating antibody specificity is essential for generating reliable research data. For DTX4 antibody validation, researchers should implement the following approaches:

• Knockdown/Knockout Controls:

  • Utilize siRNA or shRNA targeting DTX4 as demonstrated in published research

  • Compare staining patterns between wild-type and DTX4-depleted samples

  • Western blot analysis following immunoprecipitation can confirm specificity

• Overexpression Systems:

  • Transfect cells with DTX4 expression constructs

  • Compare staining intensity between transfected and non-transfected cells

  • Include empty vector controls

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide (167-432AA of human DTX4)

  • Observe reduction or elimination of specific signal

• Multi-method Validation:

  • Confirm results using different detection methods (e.g., flow cytometry and Western blot)

  • Compare staining patterns with alternative DTX4 antibodies

Studies have shown that DTX4 expression varies between tissues, with higher levels detected in pancreas, testis, placenta, and spleen. Researchers should consider this expression pattern when selecting appropriate positive and negative control samples .

How can DTX4 antibody be utilized to investigate the NLRP4-DTX4-TBK1 regulatory axis?

The NLRP4-DTX4-TBK1 pathway represents a significant negative regulatory mechanism for type I interferon signaling. Researchers investigating this pathway can employ FITC-conjugated DTX4 antibodies in several strategic approaches:

• Co-localization Studies:

  • Combine DTX4-FITC antibody with differently-labeled antibodies against NLRP4 and TBK1

  • Analyze spatial relationships using confocal microscopy

  • Quantify co-localization coefficients before and after viral infection

• Protein Interaction Analysis:

  • Perform immunoprecipitation with anti-NLRP4 or anti-TBK1 antibodies

  • Detect DTX4 using the FITC-conjugated antibody in Western blot or flow cytometry

  • Study kinetics of complex formation following viral challenge

• Functional Response Monitoring:

  • Track DTX4 recruitment during viral infection time course

  • Correlate DTX4 localization with TBK1 degradation and IRF3 phosphorylation

  • Implement live-cell imaging to capture dynamic interactions

Published research has demonstrated that the interaction between NLRP4 and TBK1 increases considerably at 8-10 hours post-VSV infection, with DTX4 being recruited to mediate TBK1 ubiquitination. This temporal pattern should be considered when designing experimental timelines .

What considerations are important when examining DTX4 in antiviral immunity studies?

When investigating DTX4's role in antiviral immunity, researchers should incorporate the following methodological considerations:

• Viral Infection Models:

  • VSV-eGFP has been effectively used in published studies

  • Consider infection at various MOI (1-10) to observe dose-dependent effects

  • Include time course analysis (2-24 hours post-infection)

• Readout Parameters:

  • Monitor type I interferon production (IFN-β ELISA, RT-qPCR for IFNB mRNA)

  • Assess interferon-stimulated genes (ISGs) expression (ISG15, IFIT1, IFIT2, CCL5)

  • Evaluate viral replication using fluorescent reporters or plaque assays

• Cell Type Considerations:

  • Human cell lines (293T, THP-1) have demonstrated DTX4 functionality

  • Primary cells (PBMCs) show physiological relevance

  • Consider tissue-specific expression patterns when selecting experimental systems

• Pathway Analysis:

  • Include ISRE luciferase reporter assays to measure IRF3-dependent signaling

  • Monitor phosphorylation status of TBK1 and IRF3

  • Assess ubiquitination status of TBK1 using K48-specific antibodies

Research has shown that knockdown of NLRP4 (which employs DTX4) enhances antiviral responses and results in reduced viral infection rates in THP-1 cells (1% vs. 55.83% at MOI of 1; 11% vs. 87% at MOI of 10) . These quantitative benchmarks provide valuable reference points for experimental design.

How should researchers address technical challenges when using FITC-conjugated antibodies in multicolor experiments?

Multicolor experiments present specific technical challenges that require methodological solutions when using FITC-conjugated DTX4 antibodies:

• Spectral Overlap Considerations:

  • FITC emission spectrum (peak ~515 nm) overlaps significantly with PE (phycoerythrin)

  • Implement proper compensation controls for each fluorophore

  • Consider using spectral analyzers for complex panels

• Signal Optimization:

  • FITC has moderate brightness compared to newer fluorophores

  • Reserve FITC for targets with moderate to high expression levels

  • Optimize voltage settings on flow cytometers for maximum resolution

• Autofluorescence Management:

  • Cellular autofluorescence often occurs in the FITC channel

  • Include unstained controls for each cell type

  • Consider alternative fluorophores for highly autofluorescent samples

• Photobleaching Prevention:

  • Minimize exposure time during fluorescence microscopy

  • Utilize anti-fade mounting media

  • Collect FITC data first in sequential imaging protocols

The FITC conjugate on the DTX4 antibody has excitation/emission wavelengths of 499/515 nm and is optimally excited by the 488 nm laser line commonly available in flow cytometers and confocal microscopes .

What is the tissue expression pattern of DTX4 and how might this influence experimental design?

Understanding DTX4's tissue distribution is crucial for experimental planning. Based on published research, DTX4 shows differential expression across human tissues:

This expression pattern has several implications for experimental design:

• Positive Control Selection:

  • Pancreatic cell lines or tissue samples provide reliable positive controls

  • BxPC-3 cells (human pancreatic cells) have demonstrated detectable DTX4 expression

• Physiological Relevance:

  • Consider tissue-specific functions when interpreting results

  • Immune-related studies may benefit from focusing on spleen-derived models

  • Reproductive biology studies may consider testis or placenta models

• Knockdown Efficiency Assessment:

  • Baseline expression levels affect detectability of knockdown effects

  • Higher expression tissues may require more robust knockdown approaches

Researchers have successfully detected endogenous DTX4 protein in 293T cells, THP-1 human monocytes, and BxPC-3 human pancreatic cells, making these suitable model systems for functional studies .

What are the biochemical properties of the DTX4 antibody that researchers should consider?

The FITC-conjugated DTX4 antibody exhibits specific biochemical properties that influence its research applications:

These properties should guide experimental design decisions, particularly regarding:

• Appropriate dilution ranges for different applications

• Selection of blocking reagents to minimize background

• Compatibility with fixation and permeabilization protocols

• Potential cross-reactivity with rabbit proteins in mixed-species samples