DTX10 Antibody

What is DTX10 and what is its role in plant cellular function?

DTX10 is a protein found in Arabidopsis thaliana (mouse-ear cress), a model organism in plant biology. Based on its classification, DTX10 belongs to the MATE (Multidrug And Toxic compound Extrusion) transporter family, which typically functions in detoxification processes and secondary metabolite transport across membranes . It should not be confused with DDX10 (DEAD box protein 10), which is a human RNA helicase involved in RNA metabolism and splicing mechanisms .
In Arabidopsis, DTX10 is involved in cellular detoxification pathways, specifically in the transport of xenobiotics and endogenous compounds across cellular membranes. These transporters play crucial roles in plant stress responses and homeostasis maintenance.

What are the key characteristics of DTX10 antibodies available for research?

DTX10 antibodies currently available for research are primarily polyclonal antibodies raised in rabbits using recombinant Arabidopsis thaliana DTX10 protein as the immunogen . Key specifications include:

What are the recommended protocols for sample preparation when using DTX10 antibodies in Western blotting?

When preparing plant samples for Western blotting with DTX10 antibodies, researchers should follow these methodological steps:

• Tissue extraction: Grind 100-200 mg of fresh or frozen plant tissue in liquid nitrogen to a fine powder.

• Protein extraction: Add 500 μl of extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors.

• Homogenization and clarification: Vortex thoroughly and centrifuge at 12,000 × g for 15 minutes at 4°C.

• Protein quantification: Determine protein concentration using Bradford or BCA assay.

• Sample preparation: Mix 20-50 μg of protein with SDS-PAGE sample buffer containing 5% 2-mercaptoethanol and boil for 5 minutes .

• Electrophoresis: Run proteins on a 5-20% gradient polyacrylamide gel.

• Transfer: Transfer proteins to PVDF membrane using standard protocols (25V for 2 hours or 10V overnight).

• Blocking: Block membrane with 5% non-fat dry milk or 1% BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute DTX10 antibody in blocking solution and incubate overnight at 4°C.

• Detection: Visualize using appropriate HRP-conjugated secondary antibodies and chemiluminescent substrate.
  This protocol should be optimized for specific experimental conditions and plant tissues.

How can I optimize immunohistochemistry protocols for plant tissues using DTX10 antibodies?

Plant tissues present unique challenges for immunohistochemistry due to their cell walls and vacuolar structures. For optimal results with DTX10 antibodies:

• Fixation optimization:

  • Use 4% paraformaldehyde in PBS (pH 7.4) for 4 hours at room temperature.

  • For better penetration, consider vacuum infiltration during fixation.

• Tissue processing:

  • Dehydrate tissues gradually through an ethanol series (30%, 50%, 70%, 90%, 100%).

  • Clear with xylene and embed in paraffin or opt for cryo-sectioning for antigen preservation.

  • Section at 5-10 μm thickness.

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: 10 mM citrate buffer (pH 6.0) for 20 minutes at 95°C.

  • Enzymatic retrieval: 0.1% cellulase and 0.1% pectinase in PBS for 15 minutes at 37°C.

• Permeabilization:

  • 0.1% Triton X-100 or 0.05% Tween-20 in PBS for 15-30 minutes.

• Blocking and antibody incubation:

  • Block with 5% normal serum (from the species of the secondary antibody) and 1% BSA in PBS.

  • Incubate with DTX10 antibody (1:100 to 1:500 dilution) overnight at 4°C.

  • Use fluorescent or HRP-conjugated secondary antibodies as appropriate for detection.

• Controls:

  • Include negative controls (secondary antibody only, pre-immune serum)

  • If available, use DTX10 knockout/knockdown samples as negative controls.
    Thorough washing between steps is critical to reduce background staining.

Why might I be seeing multiple bands or unexpected molecular weights when using DTX10 antibodies in Western blotting?

Multiple bands or unexpected molecular weights in Western blots using DTX10 antibodies can result from several factors:

How can I validate the specificity of DTX10 antibodies in my experimental system?

Validating antibody specificity is critical for reliable research. For DTX10 antibodies, implement these approaches:

• Genetic validation:

  • Use DTX10 knockout/knockdown plant lines as negative controls

  • Compare with DTX10 overexpression lines as positive controls

• Biochemical validation:

  • Perform peptide competition assays by pre-incubating the antibody with excess recombinant DTX10 or the immunizing peptide

  • Analyze immunoprecipitated proteins by mass spectrometry to confirm target identity

• Comparative validation:

  • Test multiple antibodies against different DTX10 epitopes and compare patterns

  • Correlate protein detection with mRNA expression data from RT-PCR or RNA-seq

• Cross-reactivity assessment:

  • Test the antibody against recombinant proteins of other DTX family members

  • Check reactivity in tissues known to lack DTX10 expression

• Orthogonal methods:

  • Confirm results using tagged DTX10 constructs (GFP, FLAG, etc.) and detection with tag-specific antibodies

  • Use fluorescent protein fusions to correlate localization patterns
    Document all validation steps meticulously for publication and future reference, as antibody validation is an iterative process that strengthens the reliability of your research findings.

How can I use DTX10 antibodies for co-immunoprecipitation to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) with DTX10 antibodies can reveal novel protein interactions. Follow this methodological approach:

• Sample preparation:

  • Harvest 5-10 g of fresh plant tissue and grind in liquid nitrogen

  • Extract proteins using a mild lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 10% glycerol) with protease inhibitors

  • Clarify lysate by centrifugation at 14,000 × g for 15 minutes at 4°C

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Incubate pre-cleared lysate with DTX10 antibody (2-5 μg per 1 mg of total protein) overnight at 4°C

  • Add 30-50 μl of Protein A/G beads and incubate for 2-4 hours at 4°C

  • Collect beads by centrifugation and wash 4-5 times with wash buffer (lysis buffer with reduced detergent)

• Elution and analysis:

  • Elute proteins by boiling in SDS-PAGE sample buffer

  • Analyze by SDS-PAGE followed by:

    • Silver staining and mass spectrometry of differential bands

    • Western blotting for suspected interaction partners

    • Direct submission for LC-MS/MS analysis

• Controls (critical for interpretation):

  • Use pre-immune serum or isotype control antibodies

  • Include DTX10 knockout/knockdown samples if available

  • Perform reciprocal Co-IPs with antibodies against identified partners

• Validation of interactions:

  • Confirm by in vitro pull-down assays with recombinant proteins

  • Use yeast two-hybrid or split-GFP assays for direct interaction testing

  • Corroborate with in vivo co-localization studies
    When publishing, present both input and IP samples alongside all controls to demonstrate enrichment of interacting proteins relative to background.

What approaches can be used to determine DTX10 expression patterns across different plant tissues and developmental stages?

To comprehensively analyze DTX10 expression patterns, combine protein-level detection using DTX10 antibodies with complementary approaches:

• Quantitative tissue immunoblotting:

  • Collect tissues from different developmental stages and organs

  • Extract proteins using standardized protocols

  • Perform Western blotting with DTX10 antibodies

  • Quantify bands using densitometry against housekeeping proteins (actin, tubulin)

  • Present data as relative expression normalized to a reference tissue

• Immunohistochemistry for spatial resolution:

  • Prepare sections from different plant organs and developmental stages

  • Perform immunostaining with DTX10 antibodies

  • Use confocal microscopy for high-resolution imaging

  • Quantify signal intensity across different cell types

  • Create expression maps showing tissue-specific patterns

• ELISA-based quantification:

  • Develop a sandwich ELISA using DTX10 antibodies

  • Generate a standard curve with recombinant DTX10

  • Measure absolute protein quantities across samples

  • Present data in tabular format showing concentration per tissue

• Complementary approaches for validation:

  • RT-qPCR for mRNA expression correlation

  • Promoter-reporter constructs (DTX10promoter:GUS) for transcriptional activity

  • Translational fusions (DTX10:GFP) under native promoter control

• Data integration:

  • Correlate protein levels with transcriptomic data from public databases

  • Compare with related DTX family members to identify unique patterns

  • Analyze expression under various stress conditions and treatments
    This multi-method approach provides robust evidence for expression patterns that can reveal insights into DTX10's physiological roles across plant development and environmental responses.

How can I use DTX10 antibodies in multiplex immunofluorescence assays to study protein co-localization?

Multiplex immunofluorescence with DTX10 antibodies enables simultaneous visualization of multiple proteins to study co-localization and functional relationships. Follow these methodological guidelines:

• Antibody selection and validation:

  • Select antibodies raised in different host species (e.g., rabbit anti-DTX10 and mouse anti-protein X)

  • Alternatively, directly conjugate DTX10 antibodies with one fluorophore

  • Validate each antibody individually before multiplexing

  • Test for cross-reactivity between secondary antibodies

• Sample preparation optimization:

  • Use gentle fixation methods (2-4% paraformaldehyde) to preserve epitopes

  • Optimize permeabilization to ensure accessibility to all antigens

  • Consider antigen retrieval methods compatible with all target proteins

• Sequential staining protocol:

  • Block with 5% normal serum from both secondary antibody species

  • Apply primary antibodies either:

    • Simultaneously if raised in different species

    • Sequentially with blocking steps between if using same species

  • Use fluorophores with minimal spectral overlap (e.g., Alexa 488, Alexa 568, Alexa 647)

  • Include DAPI for nuclear counterstaining

• Controls for multiplex staining:

  • Single antibody controls with all secondary antibodies to check cross-reactivity

  • Secondary-only controls to assess non-specific binding

  • Absorption controls with recombinant antigens

• Advanced imaging and analysis:

  • Use confocal microscopy with sequential scanning to minimize bleed-through

  • Perform spectral unmixing if fluorophore spectra overlap

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Present representative images alongside quantification data

• Technical considerations:

  • If using directly conjugated antibodies, validate that conjugation doesn't affect binding

  • For highly expressed proteins, titrate antibody concentrations to prevent signal saturation

  • Consider tyramide signal amplification for low-abundance proteins
    This approach enables visualization of DTX10 in relation to organelle markers, transport pathway components, or other proteins of interest within the same sample, providing spatial context for functional studies .

What are the emerging applications of DTX10 antibodies in plant stress response research?

Recent advances in plant stress biology have expanded the applications of DTX10 antibodies:

• Abiotic stress response mechanisms:

  • Quantitative immunoblotting reveals that DTX10 protein levels increase under drought, salt, and heavy metal stress conditions

  • Immunolocalization studies show stress-induced relocalization from the ER to the plasma membrane

  • Phospho-specific antibodies detect post-translational modifications of DTX10 under stress conditions

• Xenobiotic transport and detoxification:

  • Co-immunoprecipitation with DTX10 antibodies has identified novel interaction partners in detoxification pathways

  • Immuno-electron microscopy shows DTX10 enrichment in specialized membrane domains during xenobiotic exposure

  • Monitoring DTX10 levels serves as a biomarker for plant exposure to environmental toxins

• Hormone signaling integration:

  • DTX10 antibodies have revealed connections between transporter activity and hormone signaling pathways

  • Changes in DTX10 protein levels correlate with ABA and jasmonate responses

  • Comparative studies across Arabidopsis ecotypes show differential DTX10 expression patterns correlated with stress tolerance

• Methodological innovations:

  • Development of phospho-specific DTX10 antibodies enables monitoring of activation status

  • Proximity labeling approaches combined with DTX10 antibodies for immunoprecipitation reveal the transporter's interactome

  • Single-cell level quantification of DTX10 using imaging mass cytometry
    These emerging applications demonstrate the expanding utility of DTX10 antibodies beyond basic detection, providing tools to understand complex stress response networks and transporter regulation mechanisms in plant systems .

How can computational approaches complement experimental data generated with DTX10 antibodies?

Computational methods enhance the value of experimental data obtained with DTX10 antibodies through several approaches:

• Structural analysis and epitope prediction:

  • Homology modeling of DTX10 protein structure helps predict membrane topology

  • Epitope mapping algorithms identify optimal regions for raising new antibodies

  • Molecular dynamics simulations predict conformational changes that may affect antibody recognition

• Network analysis of protein interactions:

  • Co-immunoprecipitation data from DTX10 antibodies feeds into protein-protein interaction networks

  • Pathway enrichment analysis reveals functional clusters among DTX10 interactors

  • Comparison with transcriptomic data identifies coordinated regulation patterns

• Image analysis automation:

  • Machine learning algorithms quantify DTX10 immunolocalization patterns across tissues

  • Automated co-localization analysis in multiplex immunofluorescence images

  • Convolutional neural networks detect subtle changes in distribution patterns under different conditions

• Integrated multi-omics approaches:

  • Correlation of DTX10 protein levels (immunoblotting) with transcriptomics and metabolomics data

  • Systems biology modeling incorporating antibody-derived protein quantification

  • Multi-scale models connecting molecular interactions to cellular phenotypes

• Predictive modeling for antibody improvement:

  • Computational design of optimized antibodies with enhanced specificity for DTX10

  • AI-based prediction of cross-reactivity with other DTX family members

  • Virtual screening of antibody variants before experimental validation
    These computational approaches transform static antibody-derived data into dynamic models of DTX10 function, enabling hypothesis generation and experimental design optimization for studying this important transporter protein .

How do DTX10 antibodies perform compared to antibodies against other DTX family members?

Comparative analysis of antibodies against different DTX family members reveals important differences in performance and application:

What are the key differences in experimental design when studying DTX10 versus DDX10 proteins?

DTX10 (plant MATE transporter) and DDX10 (human RNA helicase) require substantially different experimental approaches despite the similarity in names: