DTX22 Antibody

What is DTX2 protein and what is its biological function?

DTX2 (Deltex homolog 2) is a protein that functions as a probable E3 ubiquitin-protein ligase. It belongs to the Deltex family with similarity to Drosophila Deltex and is also known as RNF58 (RING finger protein 58). The protein has a calculated molecular weight of approximately 67 kDa and plays important roles in signaling pathways, particularly those related to ubiquitination processes . DTX2 contains zinc ion binding domains and is involved in protein-protein interactions essential for cellular functions. The full-length human DTX2 protein consists of 575 amino acids and contains several functional domains including RING-finger motifs characteristic of E3 ubiquitin ligases .

What are the standard applications for DTX2 antibodies in research?

DTX2 antibodies are primarily validated for Western Blot (WB) applications, making them suitable for protein expression analysis in denatured samples . While Western blotting is the main validated application, some manufacturers may also suggest using these antibodies for immunohistochemistry (IHC) on both paraffin sections (IHC-p) and frozen sections (IHC-f), immunofluorescence detection of cell samples (IF/ICC), and ELISA for detecting antigenic peptides . Researchers should note that optimal dilutions for each application need to be determined empirically, as sensitivity can vary across different experimental conditions and sample types.

What species reactivity has been confirmed for DTX2 antibodies?

Commercial DTX2 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . Computational prediction models suggest potential cross-reactivity with additional species including pig, bovine, horse, sheep, rabbit, and dog samples, though these predicted reactivities require experimental validation before use in critical experiments . When working with species not explicitly listed in the validated reactivity profile, preliminary testing is strongly recommended to confirm antibody specificity and performance in the specific context of your experimental system.

How should researchers differentiate between polyclonal and monoclonal DTX2 antibodies for specific applications?

The choice between polyclonal and monoclonal DTX2 antibodies depends on experimental requirements:

What validation strategies confirm DTX2 antibody specificity and performance?

A robust validation strategy for DTX2 antibodies should include:

• Positive control testing: Confirmed DTX2-expressing cell lines such as HeLa, HepG2, MCF-7, and A549 cells should produce clear, specific bands at the expected molecular weight (67 kDa) .

• Knockout/knockdown verification: Testing antibody reactivity in DTX2 knockout or knockdown models to confirm signal extinction or reduction.

• Immunoprecipitation followed by mass spectrometry: To confirm the identity of the captured protein.

• Epitope mapping: Understanding the specific region recognized by the antibody helps predict potential cross-reactivity and effectiveness in different applications.

• Cross-reactivity assessment: Testing against related proteins (e.g., other DTX family members) to ensure specificity.

• Multiple application validation: If claiming utility across applications (WB, IHC, IF), the antibody should be validated in each context independently .
  These validation steps ensure experimental reliability and help troubleshoot unexpected results when working with DTX2 antibodies in research settings.

What is the recommended Western blot protocol for optimal DTX2 detection?

For optimal DTX2 detection using Western blot, follow this methodological approach:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Recommended protein load: 20-40 μg per lane

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels (appropriate for 67 kDa proteins)

  • Transfer to PVDF membrane at 100V for 60-90 minutes or 30V overnight

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour

  • For polyclonal antibodies (e.g., DF12523): Use 1:1000 dilution

  • For monoclonal antibodies (e.g., 67209-1-Ig): Use 1:1000-1:6000 dilution

  • Incubate with primary antibody overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

  • Wash 3-5 times with TBST

• Detection:

  • Use enhanced chemiluminescence (ECL) reagent

  • Expected band at approximately 67 kDa

• Controls:

  • Positive controls: HeLa, HepG2, MCF-7, or A549 cell lysates

  • Loading control: β-actin, GAPDH, or tubulin
    This protocol should be optimized based on your specific experimental conditions and sample types. Titration of antibody concentrations is recommended to determine the optimal signal-to-noise ratio for your samples .

How can researchers troubleshoot weak or non-specific signals when using DTX2 antibodies?

When encountering weak or non-specific signals with DTX2 antibodies, consider these methodological troubleshooting steps:
For weak signals:

• Increase protein loading: Try 40-60 μg of total protein per lane

• Optimize antibody concentration: Test concentrations up to 1:500 for enhanced sensitivity

• Extend primary antibody incubation: Overnight at 4°C or 3-4 hours at room temperature

• Use more sensitive detection systems: Switch to high-sensitivity ECL substrates

• Optimize transfer conditions: Extend transfer time or use alternative buffer systems

• Reduce washing stringency: Use lower concentrations of Tween-20 (0.05% instead of 0.1%)
  For non-specific bands:

• Increase blocking stringency: Use 5% BSA instead of milk, or extend blocking time

• Optimize antibody dilution: Test higher dilutions (1:2000-1:6000) to reduce background

• Add additional washing steps: Increase number and duration of washes

• Verify sample integrity: Check for protein degradation in your samples

• Test alternative lysate preparation methods: Different detergents may improve specificity

• Pre-absorb antibody: Incubate with non-specific proteins before application
  Implementing these methodological adjustments systematically can help identify the optimal conditions for DTX2 detection in your specific experimental system.

What considerations should guide DTX2 antibody selection for co-immunoprecipitation studies?

When selecting DTX2 antibodies for co-immunoprecipitation (co-IP) studies, researchers should consider these methodological factors:

• Epitope accessibility: Choose antibodies targeting epitopes that remain accessible in native conditions. Antibodies developed against peptides might perform poorly in co-IP compared to those raised against folded domains.

• Antibody class selection: Monoclonal antibodies often provide higher specificity for co-IP, while polyclonal antibodies may capture more protein complexes due to recognition of multiple epitopes.

• Validation requirements:

  • Verify the antibody can recognize native (non-denatured) DTX2

  • Confirm successful pull-down of DTX2 protein before proceeding to interaction studies

  • Include appropriate negative controls (isotype control antibodies)

• Buffer optimization:

  • Test different lysis conditions (detergent types and concentrations)

  • Consider gentler detergents (NP-40, Triton X-100) that preserve protein-protein interactions

  • Include appropriate protease and phosphatase inhibitors

• Technical approaches:

  • Consider pre-clearing lysates to reduce non-specific binding

  • Optimize antibody amount (typically 2-5 μg per mg of total protein)

  • Test both direct binding to beads and pre-binding to protein A/G

• Confirmation strategies:

  • Validate interactions through reciprocal co-IP when possible

  • Confirm specificity through mass spectrometry analysis of immunoprecipitated complexes
    While specific IP protocols for DTX2 antibodies aren't detailed in the provided search results, these general methodological principles should guide experimental design when studying DTX2 protein interactions .

How can DTX2 antibodies be applied in studies of ubiquitination pathways?

DTX2 functions as a probable E3 ubiquitin-protein ligase, making DTX2 antibodies valuable tools for studying ubiquitination pathways through these methodological approaches:

• Detection of DTX2-mediated ubiquitination:

  • Perform ubiquitination assays by co-expressing DTX2, substrate proteins, and tagged ubiquitin

  • Immunoprecipitate potential substrates followed by DTX2 antibody detection

  • Use DTX2 antibodies to confirm the presence of DTX2 in ubiquitin-rich cellular compartments

• Investigation of DTX2 regulation:

  • Examine DTX2 expression levels under different cellular conditions using Western blot analysis

  • Monitor DTX2 subcellular localization changes using immunofluorescence

  • Assess DTX2 protein half-life and stability through cycloheximide chase experiments

• Structure-function analysis:

  • Use DTX2 antibodies recognizing specific domains to study their role in substrate recognition

  • Implement domain mapping through deletion mutants and subsequent immunodetection

  • Compare wild-type and RING domain mutants to understand catalytic requirements

• Pathway analysis:

  • Combine DTX2 antibodies with antibodies against known E2 ubiquitin-conjugating enzymes in co-IP studies

  • Investigate the relationship between DTX2 and deubiquitinating enzymes

  • Use proximity ligation assays to visualize DTX2-substrate interactions in situ
    These methodological approaches leverage DTX2 antibodies to elucidate the protein's role in ubiquitination pathways, potentially revealing novel therapeutic targets and regulatory mechanisms .

What are the considerations for using DTX2 antibodies in multiplex immunofluorescence studies?

When designing multiplex immunofluorescence experiments involving DTX2 antibodies, researchers should consider these methodological factors:

• Antibody compatibility:

  • Select DTX2 antibodies raised in different host species than other target antibodies

  • If using same-species antibodies, consider directly conjugated primary antibodies

  • Test for cross-reactivity between secondary antibodies

• Signal optimization:

  • Determine optimal dilution ranges specifically for immunofluorescence applications

  • Test different fixation methods (paraformaldehyde, methanol, or acetone) to preserve epitopes

  • Optimize antigen retrieval methods if necessary for formalin-fixed tissues

• Controls for multiplexing:

  • Include single-stained controls to assess bleed-through between channels

  • Use isotype controls to evaluate non-specific binding

  • Include biological negative controls (tissues/cells not expressing DTX2)

• Technical considerations:

  • Order antibody application based on sensitivity (typically start with weaker signals)

  • Consider sequential rather than simultaneous staining for challenging combinations

  • Implement spectral unmixing for closely overlapping fluorophores

• Validation approaches:

  • Compare staining patterns with those obtained in single-staining experiments

  • Verify subcellular localization patterns against published literature

  • Use siRNA knockdown controls to confirm specificity in multiplex contexts
    While specific immunofluorescence protocols for DTX2 are not detailed in the provided search results, these methodological principles should guide experimental design when incorporating DTX2 antibodies into multiplex studies .

How might DTX2 antibodies contribute to understanding protein-protein interaction networks?

DTX2 antibodies can be instrumental in mapping protein-protein interaction networks through these methodological approaches:

• Affinity purification coupled with mass spectrometry:

  • Use DTX2 antibodies for immunoprecipitation followed by mass spectrometry analysis

  • Implement RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) to identify protein complexes

  • Compare interaction profiles under different cellular conditions to identify context-specific interactions

• Proximity-based labeling:

  • Combine DTX2 antibodies with BioID or APEX2 proximity labeling technologies

  • Validate proximity labeling results with traditional co-IP using DTX2 antibodies

  • Use DTX2 antibodies to confirm the expression of biotinylated proteins in specific cellular compartments

• Protein microarray applications:

  • Probe protein arrays with purified DTX2 to identify novel interactions

  • Validate array-based discoveries through reciprocal immunoprecipitation with DTX2 antibodies

  • Develop quantitative interaction maps by combining multiple antibodies against DTX2 and its partners

• Structural biology integration:

  • Use DTX2 antibodies to validate interaction interfaces identified through structural studies

  • Implement antibody-based competition assays to map binding domains

  • Develop domain-specific antibodies to dissect the interaction requirements
    These methodological approaches leverage DTX2 antibodies to construct comprehensive interaction networks, providing insights into the protein's functional roles in cellular signaling and ubiquitination pathways .

What role can DTX2 antibodies play in studying post-translational modifications?

DTX2 antibodies can be valuable tools for investigating post-translational modifications (PTMs) through these methodological approaches:

• Detection of modified DTX2 forms:

  • Use general DTX2 antibodies to identify mobility shifts indicative of modifications

  • Compare migration patterns in different cell types and under various treatments

  • Combine with phosphatase or deubiquitinase treatments to confirm modification types

• PTM-specific approaches:

  • Use phospho-specific antibodies (if available) to monitor DTX2 activation states

  • Employ ubiquitin antibodies in DTX2 immunoprecipitates to assess auto-ubiquitination

  • Implement Phos-tag gels with DTX2 antibodies to resolve phosphorylated species

• Mass spectrometry integration:

  • Immunoprecipitate DTX2 under native conditions to preserve modifications

  • Process samples for mass spectrometry to identify specific modified residues

  • Validate mass spectrometry findings with targeted antibody approaches

• Functional correlation:

  • Compare PTM profiles with DTX2 enzymatic activity measurements

  • Assess how modifications affect DTX2 subcellular localization

  • Investigate the impact of PTMs on DTX2 protein-protein interactions

• Temporal dynamics:

  • Monitor modification changes during cell cycle progression

  • Assess DTX2 modifications in response to cellular stresses

  • Track the kinetics of modification/demodification following stimulation
    These methodological approaches enable researchers to understand how post-translational modifications regulate DTX2 function, potentially revealing novel mechanisms of E3 ligase regulation and substrate recognition .