DTX3 Antibody, HRP conjugated

What is DTX3 and what cellular functions does it serve?

DTX3, also known as Deltex3 or RING finger protein 154 (RNF154), is a 347 amino acid protein containing one RING-type zinc finger domain. It belongs to the Deltex family and functions as a regulator of Notch signaling pathways. DTX3 plays a dual role as both a negative and positive regulator of Notch signaling, depending on the specific developmental and cellular context .

The protein primarily functions as a homomultimer but can also form heteromultimers with other Deltex family members, enhancing its regulatory capabilities. DTX3 exhibits highest E3 ligase activity when working in conjunction with the E2 enzyme UBE2D, which underscores the importance of protein-protein interactions in its functional regulation .

What is the molecular structure and characteristics of DTX3?

DTX3 is a 38 kDa protein consisting of 347 amino acids. Its observed molecular weight in Western blot analysis corresponds to its calculated molecular weight. The protein contains:

• One RING-type zinc finger domain

• Two WWE domains critical for its function as an E3 ubiquitin ligase

• Various structural elements that facilitate interactions with other members of the Notch signaling pathway

Two isoforms of DTX3 arise from alternative splicing, contributing to the complexity of its regulatory roles in cellular signaling pathways .

What is HRP conjugation and why is it valuable for DTX3 antibodies?

Horseradish Peroxidase (HRP) conjugation involves covalently attaching the HRP enzyme to an antibody to create a detection reagent. This conjugation provides several advantages for research applications:

• Increased sensitivity through enzymatic signal amplification

• Enhanced detection capabilities in multiple assay formats (WB, IHC, ELISA)

• Improved signal-to-noise ratio in experimental readouts

• Compatibility with various detection substrates and systems

HRP conjugation to antibodies is typically achieved by linking through lysine residues on HRP because there are only six of them, and their modification doesn't adversely affect enzyme activity . Modern conjugation kits offer directional covalent bonding of HRP to antibodies at near neutral pH, allowing high conjugation efficiency with 100% antibody recovery .

What applications are validated for DTX3 antibodies and what are the recommended protocols?

DTX3 antibodies have been validated for multiple applications with specific recommendations for optimal usage:

For IHC applications, antigen retrieval is typically performed with TE buffer at pH 9.0, though citrate buffer at pH 6.0 may be used as an alternative .

How should researchers select between polyclonal and monoclonal DTX3 antibodies?

The choice between polyclonal and monoclonal DTX3 antibodies depends on experimental goals:

Polyclonal antibodies (such as 25304-1-AP):

• Recognize multiple epitopes on the DTX3 protein

• Provide stronger signals in applications where sensitivity is crucial

• Optimal for detecting low abundance proteins or denatured forms

• Better at capturing various isoforms or slightly modified forms of DTX3

Monoclonal antibodies (such as C-10):

• Recognize a single epitope with high specificity

• Provide more consistent results across experiments

• Superior for distinguishing between closely related proteins

• Preferred for quantitative applications requiring high reproducibility

For detecting novel or poorly characterized modifications of DTX3, polyclonal antibodies may be preferable. For comparative studies requiring high reproducibility, monoclonal antibodies might be more suitable .

What are the optimal storage conditions for maintaining HRP-conjugated DTX3 antibody activity?

To maintain the activity and stability of HRP-conjugated DTX3 antibodies:

• Store at -20°C for long-term storage

• For antibodies in a solution containing glycerol (such as the 25304-1-AP with 50% glycerol), aliquoting is unnecessary for -20°C storage

• Avoid repeated freeze-thaw cycles that can degrade both antibody binding capacity and HRP enzymatic activity

• Store in appropriate buffer systems (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3)

• Ensure sodium azide concentration is minimal, as it is an irreversible inhibitor of HRP

• For working solutions, store at 4°C for short-term (1-2 weeks) use

The antibody should remain stable for one year after shipment when stored properly at -20°C .

How can researchers verify the specificity of DTX3 antibodies in experimental systems?

Verifying antibody specificity is crucial for obtaining reliable results. Recommended methods include:

• Knockdown/Knockout validation: Use siRNA or CRISPR-Cas9 systems to reduce or eliminate DTX3 expression, then confirm the corresponding reduction or absence of signal with the antibody. Published research has validated DTX3 antibodies using KD/KO approaches .

• Multiple antibody approach: Use antibodies recognizing different epitopes of DTX3 to confirm consistent detection patterns.

• Antigen competition assay: Pre-incubate the antibody with purified DTX3 protein or immunogen peptide before application to demonstrate signal reduction.

• Positive and negative control tissues: Include tissues known to express (mouse kidney, ovary, testis, brain) or not express DTX3 based on published data .

• Western blot molecular weight verification: Confirm detection at the expected 38 kDa size, matching the calculated molecular weight of DTX3 .

What considerations are important when designing experiments to study DTX3's role in Notch signaling?

When investigating DTX3's role in Notch signaling, researchers should consider:

• Cell type selection: Choose cell types where Notch signaling is active and relevant to research questions (hTERT-RPE1 cells have been validated for DTX3 expression) .

• Pathway activation/inhibition: Include experimental conditions that activate or inhibit Notch signaling to observe DTX3's regulatory effects.

• Interaction partners: Consider co-immunoprecipitation studies to investigate DTX3's interactions with other Deltex family members and Notch pathway components.

• Functional assays: Include readouts for ubiquitination activity, as DTX3 functions as an E3 ubiquitin ligase, particularly in conjunction with the E2 enzyme UBE2D .

• Isoform specificity: Account for the two known isoforms of DTX3 that arise from alternative splicing, which may have distinct functional roles .

• Localization studies: Incorporate subcellular localization experiments to determine the compartmentalization of DTX3 during active Notch signaling.

How does the preparation method for HRP-conjugated antibodies affect experimental outcomes?

The method of HRP conjugation can significantly impact experimental results:

• Conjugation chemistry: Different linking chemistries can affect the orientation of the antibody and HRP molecules, potentially impacting antigen recognition and enzymatic activity.

• Molar ratio optimization: The ideal molar ratio between antibody and HRP should be between 1:4 and 1:1 (Ab:HRP). Considering molecular weights (160,000 versus 40,000), for 1 mg HRP, 1-4 mg of antibody is recommended for optimal conjugation .

• Buffer considerations: Avoid buffers containing nucleophilic components such as primary amines and thiols (e.g., thiomersal/thimerosal) as they may react with conjugation chemicals. Sodium azide should be strictly avoided as it irreversibly inhibits HRP .

• Antibody concentration: For optimal conjugation results, the antibody should be at a concentration of 0.5-5.0 mg/ml in a volume of up to 1 ml .

• Recombinant versus chemical conjugation: Recombinant production of HRP-antibody conjugates can provide more consistent stoichiometry compared to chemical conjugation methods, though both approaches have their advantages depending on the specific application .

What are common issues with HRP-conjugated DTX3 antibodies and how can they be resolved?

Common issues and their solutions include:

• High background in Western blots:

  • Increase blocking time and concentration

  • Optimize primary and secondary antibody dilutions

  • Use more stringent washing steps

  • Include detergents like Tween-20 in wash buffers

• Weak or no signal in IHC:

  • Optimize antigen retrieval methods (try both recommended options: TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Increase antibody concentration within recommended ranges

  • Extend incubation times

  • Use signal amplification systems

• Multiple bands in Western blot:

  • Verify sample preparation (complete denaturation)

  • Check for post-translational modifications of DTX3

  • Consider the presence of different isoforms or degradation products

  • Optimize antibody dilution to reduce non-specific binding

• Loss of HRP activity:

  • Avoid sodium azide in HRP-conjugated antibody storage buffers

  • Minimize exposure to strong oxidizing agents

  • Store according to recommended conditions (-20°C with 50% glycerol)

What is the step-by-step protocol for conjugating HRP to DTX3 antibodies?

A generalized protocol for HRP conjugation to DTX3 antibodies:

• Prepare the antibody:

  • Ensure the DTX3 antibody is in an amine-free buffer (HEPES, MES, MOPS, or phosphate) at pH 6.5-8.5

  • Achieve a concentration of 0.5-5.0 mg/ml in a volume up to 1 ml

  • Remove any buffer components containing thiols or high concentrations of amines

• Prepare HRP:

  • Most commercial kits provide lyophilized HRP mix ready for conjugation

  • Rehydrate according to manufacturer instructions

• Activate conjugation chemistry:

  • For thiol-based conjugation, thiolate the antibody using appropriate reagents

  • Add modifier reagent to the antibody solution (typically 1/10 volume) and mix gently

  • Add the antibody-modifier mixture to the lyophilized HRP

• Allow conjugation to proceed:

  • Typical incubation times range from 30 minutes to 3 hours at room temperature

  • Longer incubation times may be required for higher efficiency

• Quench the reaction:

  • Add quencher reagent (typically 1/10 volume of the original antibody volume)

  • Incubate for 30 minutes at room temperature

• Purify the conjugate (optional):

  • Dialysis or gel filtration can be used to remove unconjugated components

  • Many modern kits provide 100% recovery without the need for purification

Commercial kits like the LYNX Rapid HRP Antibody Conjugation Kit simplify this process with pre-measured components and optimized protocols .

How should researchers interpret conflicting results when using DTX3 antibodies across different experimental systems?

When faced with conflicting results:

• Validate antibody performance in each system:

  • Confirm specificity in each cell/tissue type using positive and negative controls

  • Verify detection of the correct molecular weight (38 kDa for DTX3)

• Consider context-dependent expression and function:

  • DTX3 has dual roles as both positive and negative regulator of Notch signaling depending on context

  • Different cell types may express different interacting partners or regulatory components

• Evaluate experimental parameters:

  • Sample preparation methods may affect epitope accessibility

  • Buffer conditions can influence antibody-antigen interactions

  • Detection systems vary in sensitivity and dynamic range

• Account for isoform differences:

  • The two isoforms of DTX3 from alternative splicing may be detected differently by antibodies targeting different epitopes

  • Check whether the antibody recognizes all known isoforms

• Consider post-translational modifications:

  • As an E3 ubiquitin ligase, DTX3 itself may undergo modifications that affect detection

  • Different experimental conditions may promote different modification states

How can HRP-conjugated DTX3 antibodies be integrated into multiplexed detection systems?

Integrating HRP-conjugated DTX3 antibodies into multiplexed detection systems:

• Sequential multiplexing:

  • Use HRP-conjugated DTX3 antibody first with chromogenic substrate

  • Inactivate HRP with hydrogen peroxide treatment

  • Apply subsequent antibodies with different detection systems

• Spectral unmixing approaches:

  • Combine HRP-conjugated DTX3 antibody with fluorophore-conjugated antibodies targeting other proteins

  • Use computational methods to separate overlapping signals

• Tyramide signal amplification (TSA):

  • Leverage HRP's ability to catalyze the deposition of tyramide-conjugated fluorophores

  • Enable sequential multi-color staining by heat-inactivating HRP between rounds

• Spatial profiling:

  • Combine with advanced imaging techniques to map DTX3 expression in relationship to other Notch pathway components

  • Correlate with functional outcomes to understand spatial regulation

What are the advantages of recombinant HRP-conjugated antibody production versus chemical conjugation?

Comparing recombinant production with chemical conjugation:

How can researchers optimize DTX3 detection in tissues with variable expression levels?

For optimizing detection across varied expression levels:

• Titrate antibody concentration:

  • Test a range of dilutions (from 1:20 to 1:10000 depending on application)

  • Optimize for each tissue type individually

• Adjust signal amplification:

  • For tissues with low expression, use polymer-based detection systems

  • For highly expressing tissues, use direct detection methods

• Optimize antigen retrieval:

  • Compare both recommended methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Adjust retrieval times based on tissue type and fixation conditions

• Consider alternative detection substrates:

  • Use high-sensitivity substrates for low-expressing tissues

  • Use substrates with broader dynamic range for comparing tissues with variable expression

• Sample preparation optimization:

  • Adjust fixation times based on tissue type

  • Optimize section thickness for balanced signal and morphology preservation