DTX1 Antibody, HRP conjugated

What is DTX1 and what analytical methods are available for its detection?

DTX1 (Protein deltex-1) functions as a ubiquitin ligase protein in vivo, mediating ubiquitination and promoting degradation of MEKK1, suggesting it may regulate the Notch pathway via ubiquitin ligase activity . Research has demonstrated that DTX1 negatively regulates Notch1 signaling, where DTX1 depletion promotes Notch activity by elevating receptor cell-surface levels . For detection and quantification, sandwich ELISA techniques utilizing biotinylated detection antibodies specific for DTX1 with Avidin-HRP conjugate offer high sensitivity, with typical detection limits around 0.078ng/mL and assay ranges of 0.156-10ng/mL . Other methodological approaches include western blotting with HRP-conjugated antibodies for direct detection, eliminating the need for secondary antibody incubation steps, and immunocytochemistry for subcellular localization studies.

What sample types can be effectively analyzed using DTX1 antibody with HRP conjugation?

HRP-conjugated DTX1 antibodies can be used across multiple sample types and experimental systems. According to validated protocols, compatible samples include serum, plasma, whole blood, cell culture supernatants, and tissue lysates . When working with complex samples, researchers should implement appropriate extraction and purification steps to minimize matrix effects. For tissue samples, proper homogenization in compatible lysis buffers containing protease inhibitors is critical for preserving DTX1 integrity. Cultured cells can be directly lysed in appropriate buffers, while biological fluids may require dilution to optimize detection within the linear range of the assay. Performance characteristics indicate reliable results with intra-assay coefficients of variation (CV) around 3.8% and inter-assay CV of approximately 7.2% .

What are the methodological considerations for optimizing DTX1 detection using HRP-conjugated antibodies?

When optimizing DTX1 detection using HRP-conjugated antibodies, researchers should consider several methodological parameters. For western blotting applications, blocking conditions significantly impact specificity - typically 5% non-fat milk or BSA in PBST provides optimal results. The antibody dilution factor must be empirically determined, with most HRP-conjugated anti-DTX1 antibodies performing optimally between 1:100 and 1:5000 dilutions depending on the manufacturer and specific application. For chemiluminescent detection, substrate selection affects sensitivity, with enhanced chemiluminescence (ECL) substrates providing detection limits in the picogram range. For immunohistochemistry applications, antigen retrieval methods should be evaluated; acetone fixation for 2 minutes followed by methanol fixation for 2 minutes has proven effective for preserving DTX1 epitopes in cellular preparations . Incubation times and temperatures also require optimization, with most protocols recommending primary antibody incubation for 1 hour at room temperature .

How can researchers validate the specificity of DTX1 antibodies in experimental systems?

Validating DTX1 antibody specificity requires multiple complementary approaches. Primary validation should include western blot analysis against recombinant DTX1 protein and cell lysates with known DTX1 expression. The detection of a single band at the expected molecular weight (approximately 60-65 kDa) provides initial confirmation of specificity. More rigorous validation involves siRNA-mediated knockdown of DTX1, where decreased signal intensity following DTX1 depletion confirms antibody specificity . For definitive validation, researchers should consider using DTX1 knockout systems, where complete absence of signal would demonstrate absolute specificity. Cross-reactivity assessment against related Deltex family proteins (DTX2, DTX3, DTX4) is essential, particularly when studying tissues with multiple Deltex isoform expression. Additionally, peptide competition assays, where pre-incubation of the antibody with immunizing peptide blocks specific binding, provide further evidence of specificity.

How can researchers utilize DTX1 antibodies to investigate its role in Notch signaling pathways?

To effectively study DTX1's role in Notch signaling, researchers should implement a multi-faceted experimental approach. Co-immunoprecipitation experiments using HRP-conjugated DTX1 antibodies can identify protein interaction partners within the Notch pathway. Evidence indicates that DTX1 negatively regulates Notch1 signaling by preventing receptor recycling via a rab4a-dependent transport route . To investigate this mechanism, pulse-chase experiments utilizing antibodies against the Notch1 extracellular domain can track receptor recycling rates under conditions of DTX1 manipulation. Researchers should employ siRNA knockdown approaches targeting DTX1, followed by quantification of Notch target gene expression using qRT-PCR or reporter assays . For visualization of DTX1-Notch interactions, proximity ligation assays or FRET approaches with appropriately labeled antibodies provide high-resolution insights. Comparative analysis of Notch1 localization in control versus DTX1-depleted cells reveals that when DTX1 is silenced, Notch1 presence in tubulovesicular compartments is markedly reduced, with corresponding increases in nuclear localization and cell-surface expression .

What methodological approaches are recommended for studying DTX1-mediated protein trafficking?

For investigating DTX1-mediated protein trafficking, researchers should employ a combination of live-cell imaging and biochemical approaches. Antibody uptake assays using recombinant antibodies against target proteins (such as Notch1) allow monitoring of internalization and recycling kinetics . For example, after a 15-minute uptake period, antibodies against Notch1 accumulate in tubulovesicular compartments where they colocalize with antibodies recognizing the Notch1 cytoplasmic tail . Pulse-chase experiments can quantify recycling rates, with evidence showing accelerated Notch1 recycling in DTX1-depleted cells compared to controls . To identify specific recycling pathways, researchers should perform siRNA-mediated depletion of trafficking regulators (e.g., rab4a for rapid recycling, rab11 for slow recycling) in combination with DTX1 manipulation . Quantitative co-localization analysis requires proper image acquisition using confocal microscopy followed by analysis with software like ImageJ using the Colocalization Threshold plugin to determine Manders overlap coefficients .

What are the optimal experimental design considerations for investigating DTX1's ubiquitin ligase activity?

Investigating DTX1's ubiquitin ligase activity requires careful experimental design. In vitro ubiquitination assays should include purified recombinant DTX1, E1 and E2 enzymes, ubiquitin (consider using tagged versions for easier detection), ATP, and potential substrate proteins. For cellular studies, researchers should construct expression vectors for wild-type DTX1 and catalytically inactive mutants (targeting the RING domain), comparing ubiquitination patterns of target proteins. To investigate substrate specificity, proximity-dependent biotin identification (BioID) or APEX2-based proximity labeling can identify proteins in close proximity to DTX1. Studies have demonstrated that DTX1 can regulate Notch1 activity independent of direct receptor ubiquitination, as shown by experiments with KR28 Notch1 (where all 28 lysine residues in the cytoplasmic tail were mutated to arginine) . This finding highlights the importance of investigating both direct and indirect substrates of DTX1 ubiquitin ligase activity. For monitoring ubiquitination dynamics, cycloheximide chase experiments can assess protein stability, while proteasome inhibitors (MG132) can confirm degradation pathways.

How can researchers effectively troubleshoot inconsistent results in DTX1 detection assays?

When troubleshooting inconsistent DTX1 detection, researchers should systematically evaluate multiple parameters. For western blotting applications, sample preparation is critical - incomplete protein denaturation can mask epitopes, while excessive heating may cause aggregation. Buffer compatibility must be assessed, as some lysis buffers contain components that interfere with antibody binding. For ELISA applications, standard curve preparation is crucial for accurate quantification - using freshly prepared standards and ensuring proper dilution series can significantly improve consistency. Sample matrix effects can be evaluated through spike recovery experiments, with acceptable recovery rates typically between 80-120% . Technical issues with HRP conjugation can manifest as reduced signal intensity over time; researchers should monitor conjugate stability through quality control experiments and consider storing antibodies with stabilizing proteins. Cross-reactivity with related proteins (DTX2, DTX3, DTX4) may occur in specific tissues, requiring validation with isoform-specific controls. Finally, batch-to-batch variability in antibody production can be assessed through reference sample testing across multiple lots.

What advanced imaging techniques are most effective for studying DTX1 subcellular dynamics?

For advanced imaging of DTX1 subcellular dynamics, researchers should consider multiple specialized techniques. Super-resolution microscopy methods such as Structured Illumination Microscopy (SIM) or Stimulated Emission Depletion (STED) microscopy can resolve DTX1-positive structures beyond the diffraction limit, revealing detailed morphology of tubulovesicular compartments. For studying protein-protein interactions, Fluorescence Resonance Energy Transfer (FRET) can detect nanometer-scale proximity between DTX1 and potential binding partners. Live-cell imaging using HaloTag or SNAP-tag fusion proteins allows real-time tracking of DTX1 trafficking. For co-localization studies, researchers should employ dual immunofluorescence labeling followed by confocal microscopy with spectral unmixing to eliminate bleed-through. Evidence shows that DTX1 colocalizes with internalized Notch1 antibodies on tubulovesicular endosomes, and similar colocalization patterns exist between Notch1 and rab4a . Quantitative analysis of such colocalization requires proper image processing, including background subtraction using a 50-pixel rolling-ball radius followed by analysis with colocalization plugins to determine overlap coefficients .

How can DTX1 antibodies be utilized to investigate endosomal trafficking and recycling pathways?

DTX1 antibodies serve as powerful tools for investigating endosomal trafficking through several methodological approaches. Immunofluorescence co-localization studies with markers of different endosomal compartments (EEA1 for early endosomes, Rab4a for recycling endosomes, Rab11 for slow recycling endosomes, LAMP1 for lysosomes) can map the distribution of DTX1 within the endocytic pathway. Research has demonstrated that DTX1 colocalizes with Notch1 and rab4a on tubulovesicular endosomes, indicating its presence in recycling compartments . For functional studies, researchers should combine immunolocalization with genetic manipulation (siRNA knockdown or overexpression) to assess how altered DTX1 levels impact trafficking dynamics. Flow cytometry-based internalization and recycling assays using labeled antibodies against transmembrane proteins can quantify trafficking kinetics in DTX1-manipulated cells. For biochemical approaches, subcellular fractionation followed by immunoblotting for DTX1 across different endosomal fractions provides complementary evidence for its distribution. When DTX1 is depleted, PI5P4Kγ redistributes from tubulovesicular endosomes to punctate structures enriched near the nucleus, which also become positive for rab4a , suggesting DTX1 plays a role in organizing specific endosomal subdomains.

What are the recommended strategies for analyzing DTX1's interactions with PI5P4Kγ and other signaling proteins?

Analyzing DTX1's interactions with signaling proteins requires a combination of biochemical, imaging, and functional approaches. Co-immunoprecipitation studies using HRP-conjugated DTX1 antibodies can identify direct protein interactions, while proximity-based labeling methods (BioID, APEX) can capture transient or weak interactions. To investigate the relationship between DTX1 and PI5P4Kγ, researchers should examine how DTX1 depletion affects PI5P4Kγ distribution. Evidence shows that in control cells, PI5P4Kγ localizes to tubulovesicular endosomes, whereas DTX1 silencing causes redistribution to punctate structures near the nucleus that are positive for rab4a . For functional analysis, researchers should perform rescue experiments where wild-type or mutant DTX1 constructs are re-expressed in DTX1-depleted cells to determine which domains are required for proper PI5P4Kγ localization. Mass spectrometry-based interaction profiling after DTX1 immunoprecipitation can identify additional binding partners. When investigating ubiquitination targets, researchers should consider using tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins followed by immunoblotting for candidate substrates or mass spectrometry for unbiased discovery.

How can quantitative methodologies be implemented to assess DTX1 expression levels across different experimental systems?

Implementing quantitative methodologies for DTX1 assessment requires selection of appropriate techniques based on experimental needs. For absolute quantification, researchers should employ sandwich ELISA assays with HRP-conjugated detection antibodies, which offer detection ranges from 0.156-10ng/mL with sensitivities around 0.078ng/mL . Western blotting with HRP-conjugated DTX1 antibodies provides semi-quantitative data when analyzed by densitometry and normalized to housekeeping proteins. For higher throughput applications, researchers can utilize automated western platforms (e.g., Wes, Jess) for improved reproducibility and quantification. Digital PCR or qRT-PCR provides complementary data on mRNA expression, though post-transcriptional regulation may lead to discrepancies between mRNA and protein levels. Flow cytometry using fluorescently labeled antibodies enables single-cell quantification in heterogeneous populations, while imaging cytometry combines this with spatial information. For tissue samples, quantitative immunohistochemistry using digital image analysis algorithms can measure expression across different regions or cell types. Standardization across experiments requires consistent use of reference samples or calibration curves.

What experimental considerations are important when studying the effects of DTX1 mutations on protein function?

When studying DTX1 mutations, researchers should implement a comprehensive experimental strategy. Structure-function analyses should focus on key domains: the RING finger domain (critical for ubiquitin ligase activity), WWE domains (protein-protein interactions), and proline-rich regions (signaling interactions). Site-directed mutagenesis targeting these domains followed by functional assays can determine their contribution to DTX1 activity. For cellular studies, CRISPR/Cas9-mediated genome editing can introduce mutations at endogenous loci, avoiding artifacts associated with overexpression. Alternatively, researchers can employ rescue experiments where mutant DTX1 constructs are expressed in DTX1-depleted backgrounds to assess functional complementation. To evaluate effects on Notch signaling, studies show that DTX1 regulates Notch1 activity independent of direct receptor ubiquitination, as demonstrated by experiments with KR28 Notch1 (lysine-to-arginine mutations) . This finding highlights the importance of investigating both direct and indirect effects of DTX1 mutations. For interaction studies, researchers should perform co-immunoprecipitation experiments with wild-type and mutant DTX1 to identify differential binding partners. Protein stability assessments using cycloheximide chase experiments can determine if mutations affect DTX1 turnover rates.

What are the critical steps for validating HRP-conjugated DTX1 antibodies before experimental use?

Comprehensive validation of HRP-conjugated DTX1 antibodies requires multiple quality control steps. Initially, researchers should verify conjugation efficiency through spectrophotometric analysis, with optimal HRP:antibody ratios typically between 2:1 and 4:1. Activity assessment using a simple dot blot or western blot with known positive controls confirms the conjugate's functionality. Specificity validation should include western blotting against recombinant DTX1 and cellular lysates with known DTX1 expression patterns, looking for a single band at approximately 65 kDa. More rigorous validation requires testing in DTX1 knockdown or knockout systems, where signal reduction or elimination confirms specificity . Cross-reactivity assessment against related Deltex family proteins (DTX2, DTX3, DTX4) is essential when working in tissues expressing multiple isoforms. Stability testing under various storage conditions helps establish optimal handling protocols - typical recommendations include storage at 4°C with glycerol or stabilizing proteins for short-term use, or aliquoting and freezing at -20°C for long-term storage. Lot-to-lot consistency should be verified using reference samples to ensure reproducible results across experiments.

How should researchers optimize immunolocalization protocols using HRP-conjugated DTX1 antibodies?

Optimizing immunolocalization with HRP-conjugated DTX1 antibodies requires careful attention to multiple experimental parameters. Fixation method significantly impacts epitope preservation and accessibility - research protocols have successfully used ice-cold acetone for 2 minutes followed by methanol for 2 minutes for optimal DTX1 detection . Permeabilization conditions must be optimized based on the subcellular compartment of interest; 0.1% Tween in PBS (PBST) provides suitable permeabilization for most applications . Blocking protocols typically require 5-10% normal serum matching the species of the secondary antibody, or BSA to minimize background. Antibody dilution optimization should follow a titration approach, typically testing ranges from 1:50 to 1:500 for primary antibodies. For HRP-conjugated antibodies, direct detection using appropriate substrates (DAB for brightfield, tyramide signal amplification for fluorescence) eliminates the need for secondary antibodies. Incubation conditions typically involve 1 hour at room temperature, though overnight incubations at 4°C may enhance sensitivity for low-abundance targets . Signal development timing is critical - over-development leads to high background, while under-development results in weak signals. For co-localization studies, sequential detection with different chromogens or fluorophores allows visualization of multiple targets.

What are the recommended approaches for quantifying western blot results using HRP-conjugated DTX1 antibodies?

Quantifying western blot results with HRP-conjugated DTX1 antibodies requires systematic approaches to ensure accuracy and reproducibility. Sample preparation standardization is fundamental - consistent cell lysis conditions, protein determination methods, and loading amounts (typically 10-30 μg total protein) minimize variation. For electrophoresis, gradient gels (4-12% or 4-20%) often provide optimal resolution of DTX1 at approximately 65 kDa. Transfer efficiency should be verified using reversible staining methods like Ponceau S before immunodetection. When using HRP-conjugated primary antibodies, researchers should optimize dilution factors, typically between 1:1000 and 1:5000, and minimize washing steps to prevent signal loss. Chemiluminescent substrate selection affects sensitivity and dynamic range - enhanced chemiluminescence substrates provide sensitive detection, while newer technologies like SuperSignal West Pico PLUS offer expanded dynamic ranges suitable for quantification. Image acquisition must use systems with linear detection ranges (CCD cameras rather than film) and avoid saturation. Densitometric analysis should include background subtraction, normalization to loading controls (preferably from the same membrane), and calculation of relative expression levels. Multiple biological and technical replicates (minimum n=3) are essential for statistical validity, with results typically reported as mean ± standard deviation.

What quality control measures should be implemented when performing ELISA assays with DTX1 antibodies?

ELISA assays with DTX1 antibodies require comprehensive quality control measures throughout the workflow. Standard curve preparation is foundational - using recombinant DTX1 protein standards with verified purity (≥95%) and creating fresh standard dilutions for each assay ensures accuracy. The standard curve should include at least 7 points with duplicate or triplicate measurements and demonstrate linearity across the expected sample concentration range (typically 0.156-10 ng/mL) . Sample preparation protocols must be standardized, including consistent collection, processing, and storage conditions to minimize pre-analytical variability. For each assay, researchers should include positive and negative controls - these might involve samples with known DTX1 concentrations or biological samples with verified high or low expression. Technical controls should include blank wells (no sample or standard) to assess background signal and substrate controls (all reagents except detection antibody) to evaluate non-specific enzyme activity. Assay performance is typically evaluated through intra-assay CV (<10%, ideally <5%) and inter-assay CV (<15%, ideally <10%) . Spike recovery experiments, where known amounts of recombinant DTX1 are added to sample matrix, should yield 80-120% recovery rates. Parallelism testing, where serial dilutions of samples show response curves parallel to the standard curve, confirms absence of matrix effects.

How are DTX1 antibodies being used to investigate novel signaling pathways beyond Notch?

DTX1 antibodies are increasingly employed to explore signaling networks beyond the canonical Notch pathway. While DTX1 is well-established as a negative regulator of Notch1 signaling , recent research reveals its involvement in additional signaling cascades. Proteomic approaches using HRP-conjugated DTX1 antibodies for immunoprecipitation followed by mass spectrometry have identified novel interaction partners. Evidence suggests DTX1 functions as a ubiquitin ligase protein mediating ubiquitination and degradation of MEKK1, potentially regulating MAPK signaling pathways . For investigating these interactions, researchers should implement proximity-dependent labeling methods (BioID, APEX) to capture the broader DTX1 interactome. ChIP-seq approaches can identify potential transcriptional programs regulated by DTX1, while phospho-proteomic analysis before and after DTX1 depletion can reveal effects on kinase signaling networks. To study DTX1's involvement in trafficking of receptors beyond Notch, antibody uptake and recycling assays can be applied to RTKs, GPCRs, and other transmembrane signaling proteins. Researchers should combine these approaches with functional readouts specific to different signaling pathways to comprehensively map DTX1's regulatory role across cellular signaling networks.

What methodological advances are improving the study of DTX1 in primary tissues and complex biological systems?

Methodological advances enhancing DTX1 research in complex systems span multiple technological domains. For tissue analysis, multiplex immunofluorescence techniques now allow simultaneous visualization of DTX1 alongside multiple markers, providing contextual information about expressing cell types and their microenvironment. Tissue clearing methods (CLARITY, CUBIC, iDISCO) combined with light-sheet microscopy enable 3D visualization of DTX1 distribution throughout intact tissues. At the single-cell level, mass cytometry (CyTOF) using metal-conjugated DTX1 antibodies can quantify expression across heterogeneous populations while simultaneously measuring dozens of other parameters. Spatial transcriptomics technologies can correlate DTX1 protein expression with local transcriptional programs in tissue sections. For functional studies in primary cells, improved transduction methods using lentiviral or AAV vectors allow efficient genetic manipulation of DTX1 in hard-to-transfect primary cells. Organoid systems derived from primary tissues provide physiologically relevant models for studying DTX1 function in 3D contexts that better recapitulate tissue architecture. Patient-derived xenografts (PDX) enable investigation of DTX1 function in human disease models while maintaining tumor heterogeneity and microenvironment complexity.