DTX28 Antibody

What is DTX28 and why are antibodies developed against it?

DTX28 is a protein found in Arabidopsis thaliana (Mouse-ear cress), a widely used model organism in plant biology. Antibodies against DTX28 are developed to study its expression, localization, and function in plant systems. These antibodies serve as valuable tools for understanding various cellular processes and pathways in which DTX28 is involved. Based on the naming convention, DTX28 likely belongs to the DETOXIFICATION (DTX) protein family, which includes transporters involved in detoxification processes in plants .

What are the primary applications of DTX28 antibodies in plant research?

DTX28 antibodies are primarily used in:

• Western Blot (WB) analysis to detect and quantify DTX28 protein expression

• ELISA assays for protein quantification

• Immunohistochemistry (IHC) to visualize protein localization in plant tissues

• Co-immunoprecipitation experiments to study protein-protein interactions
  These applications enable researchers to investigate DTX28's role in plant development, stress responses, and other physiological processes .

What validation methods are recommended for DTX28 antibodies?

Proper validation of DTX28 antibodies is crucial for reliable experimental results. Recommended validation methods include:

How should I design experiments to study DTX28 protein expression across different plant tissues?

When studying DTX28 expression across plant tissues:

• Select tissues representing various developmental stages and conditions

• Prepare consistent protein extracts with appropriate extraction buffers containing protease inhibitors

• Normalize protein loading (typically 50 μg per lane for Western blot)

• Use validated DTX28 antibody at the recommended dilution (typically 1:1000-1:6000 for Western blot)

• Include appropriate controls (positive protein control, negative control, loading control)

• Quantify expression using densitometry and normalize to a housekeeping protein
  For tissue-specific localization, immunohistochemistry on fixed tissue sections can be performed using DTX28 antibody followed by a compatible secondary antibody system .

What are the optimal storage conditions for maintaining DTX28 antibody activity?

Based on manufacturer recommendations for similar antibodies, DTX28 antibodies should be stored as follows:

How can I use DTX28 antibodies to investigate protein-protein interactions in plant stress response pathways?

To investigate DTX28's protein interactions during stress responses:

• Co-immunoprecipitation approach:

  • Treat plants with relevant stress conditions (e.g., drought, salt, pathogen)

  • Prepare native protein extracts using gentle detergents (0.1% Triton X-100)

  • Immunoprecipitate with DTX28 antibody bound to Protein A/G beads

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm interactions with reverse co-IP using antibodies against identified partners

• Proximity labeling approach:

  • Generate fusion proteins with DTX28 and BioID or APEX2

  • Express in plant systems and activate labeling during stress conditions

  • Purify biotinylated proteins and identify by mass spectrometry
    These approaches have been successfully used to study protein interactions in similar plant stress response pathways .

What methodologies are recommended for mapping the epitope recognized by DTX28 antibodies?

For epitope mapping of DTX28 antibodies, consider these advanced methodologies:

• Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS):

  • Incubate DTX28 protein with and without antibody

  • Perform hydrogen-deuterium exchange

  • Digest with pepsin and analyze peptide fragments by MS

  • Identify regions protected from exchange when antibody is bound

• Peptide array analysis:

  • Generate overlapping peptides spanning the DTX28 sequence

  • Test antibody binding to peptide arrays

  • Identify peptides with highest binding affinity

• Alanine scanning mutagenesis:

  • Create point mutations in recombinant DTX28

  • Test antibody binding to mutants

  • Identify critical residues for antibody recognition
    HDX-MS has been particularly effective for epitope mapping of various antibodies, providing high-resolution structural information about antibody-antigen interactions .

What strategies can resolve non-specific binding issues with DTX28 antibodies in Western blot applications?

To improve specificity in Western blot applications:

How can researchers address cross-reactivity between DTX28 and other DTX family members?

Cross-reactivity with other DTX family proteins is a significant challenge. To address this:

• Perform sequence alignment analysis:

  • Align DTX28 with other DTX family proteins to identify unique regions

  • Design peptide competition assays using unique and shared sequence regions

• Validation in knockout/knockdown systems:

  • Test antibody in DTX28 knockout/knockdown plants

  • All signals should be reduced/eliminated in true DTX28-specific antibodies

• Differential expression analysis:

  • Compare antibody staining patterns with known tissue-specific expression of DTX family members

  • Discrepancies may indicate cross-reactivity

• Immunoprecipitation-Mass Spectrometry:

  • Immunoprecipitate with the DTX28 antibody

  • Identify captured proteins by mass spectrometry to assess specificity
    If cross-reactivity is confirmed, consider generating new antibodies against unique DTX28 epitopes or using epitope-tagged DTX28 in transgenic systems .

How can DTX28 antibodies be integrated into advanced chromatin immunoprecipitation (ChIP) studies?

For researchers investigating DTX28's potential role in chromatin regulation:

• ChIP-seq protocol optimization:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with DTX28 antibody (use 5-10 μg per reaction)

  • Prepare libraries for next-generation sequencing

  • Analyze enriched genomic regions to identify potential DNA binding sites

• CUT&RUN adaptations for plant systems:

  • Isolate nuclei from plant tissue

  • Bind DTX28 antibody to isolated nuclei

  • Target MNase digestion to antibody-bound regions

  • Isolate released DNA fragments for sequencing
    These approaches have been successfully used to study chromatin-associated proteins in Arabidopsis, as demonstrated in research on SWR1 complex components that used similar antibody-based techniques .

What are the latest methodologies for using DTX28 antibodies in single-cell protein analysis in plant tissues?

Emerging technologies for single-cell protein analysis using DTX28 antibodies include:

• Mass cytometry (CyTOF) adaptations for plant cells:

  • Develop metal-conjugated DTX28 antibodies

  • Optimize plant cell preparation protocols

  • Analyze protein expression at single-cell resolution

• Imaging mass cytometry:

  • Apply metal-labeled DTX28 antibodies to tissue sections

  • Perform laser ablation and mass spectrometry

  • Create high-resolution spatial maps of protein expression

• Proximity ligation assays (PLA):

  • Use DTX28 antibody with antibodies against potential interacting partners

  • Visualize protein-protein interactions in situ with single-molecule resolution

  • Quantify interaction frequency across different cell types
    While these techniques are still being adapted for plant systems, they represent promising directions for understanding DTX28 function at cellular and subcellular resolution .

How can researchers correlate DTX28 protein levels with transcriptomic data in stress response studies?

To integrate protein-level data with transcriptomics:

• Experimental design considerations:

  • Collect samples for both protein and RNA analysis from the same tissue

  • Include multiple timepoints to capture expression dynamics

  • Apply stress treatments consistently across experimental replicates

• Quantitative Western blot methodology:

  • Use DTX28 antibody with fluorescently labeled secondary antibodies

  • Include recombinant DTX28 protein standards for absolute quantification

  • Normalize to housekeeping proteins that remain stable under stress conditions

• Data integration approach:

  • Plot normalized DTX28 protein levels against mRNA expression values

  • Calculate correlation coefficients and time delays between transcript and protein changes

  • Apply regression analysis to model the relationship between transcription and translation
    This integrated approach can reveal post-transcriptional regulation mechanisms affecting DTX28 expression during stress responses .

What methodologies can combine DTX28 localization studies with functional assays in planta?

To correlate DTX28 localization with function:

• Combined fluorescence approaches:

  • Perform immunofluorescence with DTX28 antibodies

  • Simultaneously visualize cellular markers or physiological parameters (e.g., ROS indicators, ion-sensitive fluorophores)

  • Analyze colocalization and temporal relationships

• Correlative light and electron microscopy:

  • Detect DTX28 using immunogold labeling for electron microscopy

  • Correlate with live-cell imaging using fluorescent markers

  • Generate high-resolution maps of DTX28 localization relative to cellular structures

• Functional manipulation with spatiotemporal precision:

  • Use optogenetic tools to manipulate DTX28 activity in specific subcellular compartments

  • Monitor resultant physiological changes in real-time

  • Correlate with DTX28 localization determined by immunofluorescence
    These approaches enable researchers to move beyond correlative observations to establish causal relationships between DTX28 localization and function .

How might computational approaches enhance the development of next-generation DTX28 antibodies?

Advanced computational methods show promise for DTX28 antibody development:

• In silico epitope prediction:

  • Analyze DTX28 structure using AlphaFold2 or similar algorithms

  • Predict surface-exposed regions with high antigenicity

  • Identify epitopes that distinguish DTX28 from related proteins

• Antibody design using machine learning:

  • Train models on successful plant antibody datasets

  • Generate optimized antibody variable regions in silico

  • Design complementarity-determining regions (CDRs) with high specificity for DTX28

• Molecular dynamics simulations:

  • Model antibody-antigen interactions at atomic resolution

  • Optimize binding affinity through targeted mutations

  • Predict cross-reactivity with related proteins
    These computational approaches, combined with experimental validation, could significantly accelerate the development of highly specific DTX28 antibodies with enhanced performance characteristics .

What emerging technologies might transform how researchers apply DTX28 antibodies in plant science?

Emerging technologies with potential to revolutionize DTX28 antibody applications include:

• Nanobody engineering:

  • Develop single-domain antibodies (nanobodies) against DTX28

  • Express directly in plant cells for intracellular immunomodulation

  • Combine with fluorescent proteins for live-cell tracking

• CRISPR-based protein tagging:

  • Engineer epitope tags into endogenous DTX28 locus

  • Use well-characterized antibodies against the tag

  • Maintain native expression patterns and regulation

• Spatially-resolved proteomics:

  • Apply DTX28 antibodies in spatial transcriptomics platforms

  • Create cell-type-specific protein expression maps

  • Correlate with single-cell RNA sequencing data

• Antibody-based biosensors:

  • Develop FRET-based sensors using DTX28 antibody fragments

  • Monitor protein conformation changes in real-time

  • Detect post-translational modifications in living cells
    These approaches represent the frontier of plant molecular biology and offer new ways to understand DTX28 function in complex biological contexts .

What is a recommended comprehensive workflow for DTX28 antibody validation and application?

A best-practice workflow for DTX28 antibody research involves:

How should researchers select the most appropriate DTX28 antibody for their specific experimental goals?

When selecting a DTX28 antibody, consider these factors based on your experimental needs:

• For protein detection and quantification:

  • Select antibodies validated for Western blot

  • Consider sensitivity requirements (monoclonal antibodies often provide better quantitative results)

  • Check reported detection limits in literature

• For protein localization:

  • Choose antibodies validated for immunohistochemistry/immunofluorescence

  • Verify performance in fixed plant tissues

  • Consider species cross-reactivity if working with non-Arabidopsis plants

• For protein-protein interaction studies:

  • Select antibodies validated for immunoprecipitation

  • Consider potential epitope masking in protein complexes

  • Test for interference with known interaction domains

• For multiple applications:

  • Polyclonal antibodies often work across multiple applications

  • Check validation data for each intended application

  • Consider acquiring application-specific antibodies if needed
    Always review the full validation data and literature citations before selecting an antibody for your research .