DTX3 Antibody, Biotin conjugated

What is DTX3 and what cellular functions does it regulate?

DTX3 (Deltex Homolog 3) is a 347 amino acid protein that contains one RING-type zinc finger domain and belongs to the Deltex protein family. It functions as an E3 ubiquitin ligase protein in vitro, suggesting it may regulate the Notch signaling pathway through ubiquitin ligase activity .

The Notch signaling pathway is critically involved in cell-cell communications that regulate a broad spectrum of cell-fate determinations. DTX3 acts as a key regulator in this pathway, influencing cellular differentiation and developmental processes. With an observed molecular weight of approximately 38 kDa, DTX3 plays important roles in regulating cellular processes through its interaction with the Notch pathway components .

What is the structural composition of a biotin-conjugated DTX3 antibody?

A biotin-conjugated DTX3 antibody consists of an anti-DTX3 antibody molecule with biotin molecules covalently attached to it through a conjugation process. In typical commercial preparations like the documented anti-DTX3 antibody (AA 90-347), the antibody is raised against specific epitopes (amino acids 90-347) of the human DTX3 protein .

The biotinylation process typically achieves a controlled biotin:antibody molar ratio, which is crucial for maintaining antibody functionality while providing sufficient biotin molecules for detection. For instance, in similar biotinylated antibody systems, an optimal average molar biotin:antibody ratio ranges from 2.5 to 4.0, as seen in other biotinylated monoclonal antibodies . This ratio ensures the antibody retains its immunocompetence while providing sufficient biotin molecules for avidin/streptavidin binding.

How stable is biotin-conjugated DTX3 antibody, and what are the recommended storage conditions?

Biotin-conjugated antibodies, including DTX3 antibodies, are typically stored in buffers containing preservatives like ProClin 300 (0.03%) and stabilizers such as glycerol (50%) in PBS at pH 7.4 . For long-term stability, these antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment .

For optimal preservation of activity, it's advisable to:

• Avoid repeated freeze-thaw cycles

• Store in small aliquots if frequent use is anticipated

• Keep protected from light, especially if other fluorescent tags are present

• Avoid prolonged exposure to room temperature

Some preparations may contain additional stabilizers like bovine serum albumin (BSA) at concentrations of approximately 10 mg/mL to maintain antibody integrity and prevent non-specific binding .

What are the primary applications for biotin-conjugated DTX3 antibodies in research?

Biotin-conjugated DTX3 antibodies are valuable tools in multiple research applications, primarily:

• ELISA: The biotin-conjugated format is particularly suitable for enzyme-linked immunosorbent assays, where the biotin tag enables sensitive detection through streptavidin-enzyme conjugates .

• Immunohistochemistry (IHC): While the biotin-conjugated format may be used directly, it's also employed in multi-step detection systems leveraging the high-affinity biotin-avidin interaction .

• Western Blotting: For detecting DTX3 protein in complex samples with high specificity. The biotin conjugation allows for flexible detection systems and potential signal amplification .

• Flow Cytometry: Similar to other biotin-conjugated antibodies like CD3 antibodies, DTX3 biotin conjugates can be used in multi-color flow cytometry applications when combined with appropriate streptavidin-fluorophore conjugates .

• Pretargeting Applications: In advanced research settings, biotinylated antibodies are used in three-step pretargeting protocols where unlabeled biotinylated antibodies are administered first, followed by clearing agents and radiolabeled biotin for imaging or therapeutic applications .

How should I optimize the three-step pretargeting protocol when using biotinylated antibodies like DTX3 for imaging applications?

The three-step pretargeting protocol requires careful optimization of several parameters to achieve high target-to-background ratios. Based on studies with biotinylated monoclonal antibodies, the following methodological approach is recommended:

• First step - Biotinylated antibody administration:

  • Use purified biotinylated antibody with a biotin:antibody ratio of 2.0-3.0

  • Administer intravenously and allow sufficient time (typically 24-48 hours) for binding to target and clearance from circulation

• Second step - Clearing agent:

  • Administer avidin or streptavidin (typically 50-200 μg) intravenously

  • This clears circulating biotinylated antibody while leaving target-bound antibody accessible

• Third step - Radiolabeled biotin:

  • Administer DTPA-biotin labeled with an appropriate radioisotope (e.g., 111In or 99mTc)

  • Optimal timing between clearing agent and radiolabeled biotin administration is crucial

When optimized, studies have shown tumor/blood ratios can increase from 1.5 to 4.0 by extending the interval between streptavidin and radiolabeled biotin injections from 1 day to 7 days .

The typical biodistribution pattern shows rapid clearance of radiolabeled biotin from blood and non-target tissues, with %ID/g values in blood reduced significantly (p=0.009) after 1 hour, as shown in the following excerpt from biodistribution data:

Adapted from biodistribution studies

What is the recommended protocol for conjugating biotin to DTX3 antibodies while preserving antibody functionality?

While specific protocols for DTX3 antibody biotinylation aren't detailed in the provided sources, general methodological principles for antibody biotinylation that maintain functionality include:

• Selection of appropriate biotinylation reagent:

  • NHS-LC-biotin (N-hydroxysuccinimide-long chain-biotin) is commonly used for primary amine conjugation

  • For controlled conjugation, use a biotin:antibody molar ratio of 5:1 to 20:1 depending on desired final conjugation level

• Conjugation procedure:

  • Perform conjugation in slightly alkaline conditions (pH 7.4-8.2) to facilitate NHS ester reaction with primary amines

  • Maintain antibody concentration at 1-5 mg/mL during conjugation

  • React for 30-60 minutes at room temperature or 2 hours at 4°C

• Purification:

  • Remove unreacted biotin using size exclusion chromatography or dialysis

  • Analyze the conjugate by size exclusion HPLC to confirm monomer content and absence of aggregates

• Verification:

  • Determine the biotin:antibody ratio using HABA assay or mass spectrometry

  • Confirm immunocompetence through binding assays (e.g., ELISA, flow cytometry)

  • Target an average of 2-4 biotin molecules per antibody for optimal performance

The conjugation should yield antibodies with approximately 90% immunocompetent fraction and titer values comparable to unconjugated antibodies (e.g., 1:3,200 as observed with other biotinylated monoclonal antibodies) .

How specific are biotin-conjugated DTX3 antibodies across different species and what cross-reactivity can be expected?

Based on available data for DTX3 antibodies, specificity varies by antibody clone and preparation. For the biotinylated DTX3 antibody (AA 90-347), testing indicates:

• Species reactivity:

  • Confirmed reactivity with human DTX3

  • Potential cross-reactivity with mouse DTX3 has been observed with some DTX3 antibody clones

• Cross-reactivity considerations:

  • DTX3 belongs to the Deltex family, which includes DTX1, DTX2, and DTX4 homologs

  • Epitope selection (e.g., AA 90-347) helps minimize cross-reactivity with other Deltex family members

  • Different regions of DTX3 show varying conservation across species, with the RING-finger domain being most conserved

When conducting experiments across species, it's advised to first validate the antibody in the target species via western blot or immunohistochemistry. Published studies have confirmed reactivity of some DTX3 antibodies with tissues including:

• Human kidney and colon tissues (via IHC)

• Mouse kidney, ovary, testis, and brain tissues (via WB)

• Multiple human and mouse cell lines including hTERT-RPE1 cells

How can I confirm the specificity of my biotin-conjugated DTX3 antibody before using it in critical experiments?

To validate the specificity of biotin-conjugated DTX3 antibodies before conducting critical experiments, implement this systematic approach:

• Positive and negative control tissues/cell lines:

  • Use tissues with known DTX3 expression as positive controls (e.g., mouse testis, kidney, brain)

  • Include tissues or cell lines with confirmed low/no DTX3 expression as negative controls

  • For human samples, kidney and colon tissues have shown positive DTX3 expression

• Blocking experiments:

  • Pre-incubate the antibody with recombinant DTX3 protein (preferably the immunogen)

  • Compare staining patterns between blocked and unblocked antibody

  • Specific binding should be significantly reduced in blocked samples

• Knockout/knockdown validation:

  • Test the antibody on samples from DTX3 knockout models or cells with DTX3 knockdown

  • Compare with wild-type samples to confirm specificity

  • Published knockdown studies have used DTX3 antibodies for validation

• Western blot molecular weight verification:

  • Confirm detection of a band at the expected molecular weight (~38 kDa for DTX3)

  • Check for absence of unexpected bands that might indicate cross-reactivity

  • Use reducing and non-reducing conditions to assess antibody performance

• Comparative analysis with alternative antibody clones:

  • Test multiple antibodies targeting different epitopes of DTX3

  • Concordant results across different antibodies increase confidence in specificity

This validation framework ensures experimental reliability and data integrity, particularly important for biotin-conjugated antibodies where avidin/streptavidin systems can sometimes contribute to background signals.

What are common causes of high background when using biotin-conjugated DTX3 antibodies, and how can they be addressed?

High background is a common challenge when using biotin-conjugated antibodies, including those targeting DTX3. The primary causes and their solutions include:

• Endogenous biotin interference:

  • Cause: Many tissues (especially liver, kidney, and brain) contain endogenous biotin that can bind directly to detection reagents

  • Solution: Implement biotin blocking steps prior to primary antibody incubation using commercial biotin blocking kits or sequential avidin and biotin blocking

• Non-specific binding of the primary antibody:

  • Cause: Hydrophobic or ionic interactions between antibody and sample components

  • Solution: Optimize blocking conditions (5-10% normal serum from the same species as the secondary reagent), increase blocking time (1-2 hours), and include 0.1-0.3% Triton X-100 or Tween-20 in antibody diluent

• Excessive biotinylation of the antibody:

  • Cause: Over-biotinylated antibodies can increase non-specific binding or form aggregates

  • Solution: Use antibodies with optimal biotin:antibody ratios (typically 2-4 biotin molecules per antibody) and confirm proper purification of the conjugate

• Cross-reactivity with related proteins:

  • Cause: Antibody recognizing epitopes shared with other Deltex family members

  • Solution: Use antibodies targeting unique regions of DTX3 and validate specificity using knockout/knockdown controls

• Excessive detection reagent concentration:

  • Cause: Too much streptavidin-conjugate can increase background

  • Solution: Titrate detection reagents carefully, typically using 1:1000-1:5000 dilutions depending on the application and specific conjugate

For biotin-conjugated DTX3 antibody applications, dilution ranges of 1:2000-1:10000 for Western blot and 1:20-1:200 for IHC have been recommended for related DTX3 antibodies, which can serve as starting points for optimization .

How can I optimize the signal-to-noise ratio when using biotin-conjugated DTX3 antibodies in immunohistochemistry?

Optimizing signal-to-noise ratio for biotin-conjugated DTX3 antibodies in immunohistochemistry requires systematic protocol refinement:

• Antigen retrieval optimization:

  • For DTX3 detection, heat-induced epitope retrieval using TE buffer (pH 9.0) has shown good results

  • Alternative approach: citrate buffer (pH 6.0) can be used with certain tissue preparations

  • Test both methods to determine optimal conditions for your specific tissue

• Blocking optimization:

  • Implement dual blocking approach:
    a. Protein block: 5-10% normal serum (from same species as secondary reagent) for 30-60 minutes
    b. Biotin/avidin block: Commercial kits or sequential avidin/biotin blocking steps

• Antibody dilution optimization:

  • Create a dilution series (e.g., 1:20, 1:50, 1:100, 1:200) to determine optimal concentration

  • Starting range for DTX3 antibodies in IHC applications: 1:20-1:200

  • Extend primary antibody incubation to overnight at 4°C for increased specific signal

• Detection system considerations:

  • For low-abundance targets like DTX3, consider using amplification systems:
    a. Tyramide signal amplification (TSA)
    b. Multiple-layer streptavidin-biotin systems

  • Balance amplification with potential increased background

• Washing optimization:

  • Increase number and duration of washes (minimum 3 x 5 minutes)

  • Use 0.05-0.1% Tween-20 in wash buffer to reduce non-specific binding

  • Include additional high-salt wash step (PBS with 0.5M NaCl) to reduce ionic interactions

• Counterstain adjustment:

  • Use lighter hematoxylin counterstaining to avoid masking specific signal

  • For fluorescent detection, carefully select fluorophores to avoid tissue autofluorescence

By systematically optimizing these parameters, researchers can achieve improved signal-to-noise ratios in DTX3 immunohistochemistry staining, particularly in tissues with demonstrated DTX3 expression such as human kidney and colon tissues .

How can biotin-conjugated DTX3 antibodies be incorporated into multi-parameter analysis of Notch signaling pathway components?

Biotin-conjugated DTX3 antibodies offer unique advantages in multi-parameter analysis of Notch signaling due to their compatibility with diverse detection systems. Here's a methodological approach for integrating these antibodies into comprehensive Notch pathway analysis:

• Multi-color flow cytometry:

  • Combine biotin-conjugated DTX3 antibody with directly labeled antibodies against other Notch components (Notch receptors, ligands, MAML)

  • Use streptavidin conjugated to a spectrally compatible fluorophore for DTX3 detection

  • Implement multi-parameter compensation and analysis to correlate DTX3 with other pathway components

• Multiplex immunohistochemistry/immunofluorescence:

  • Sequential staining approach:
    a. First round: Biotin-DTX3 antibody + streptavidin-HRP + tyramide-fluorophore 1
    b. Heat denaturation or chemical stripping
    c. Second round: Next target antibody + detection system
    d. Repeat for additional targets (3-7 markers possible)

  • This approach allows visualization of DTX3 in spatial context with other Notch components

• Proximity ligation assay (PLA) for protein-protein interactions:

  • Use biotin-conjugated DTX3 antibody with streptavidin-oligonucleotide

  • Pair with antibody against potential interacting protein (e.g., other Notch pathway components)

  • Proximity-dependent signal amplification reveals direct interactions between DTX3 and other proteins

• ChIP-seq applications:

  • For examining DTX3 interactions with chromatin in Notch signaling context

  • Utilize biotin-conjugated DTX3 antibody with streptavidin magnetic beads

  • Sequence captured DNA to identify DTX3-associated genomic regions

• Multiparameter Western blotting:

  • Sequential or multiplexed detection of DTX3 and other Notch components

  • Use spectrally distinct fluorophore-conjugated streptavidin for DTX3 detection

  • Combine with directly labeled antibodies against other components on the same blot

These approaches leverage the biotin-streptavidin system's versatility while enabling comprehensive analysis of DTX3's role within the broader Notch signaling network, facilitating deeper understanding of regulatory mechanisms and potential therapeutic targets.

What are the advantages and limitations of using biotin-conjugated DTX3 antibodies compared to directly labeled fluorescent conjugates in advanced imaging applications?

Advanced imaging applications require careful consideration of detection systems. Biotin-conjugated DTX3 antibodies present distinct advantages and limitations compared to directly labeled fluorescent conjugates:

Advantages:

• Signal amplification potential:

  • Biotin-streptavidin systems allow multi-layer amplification, enhancing sensitivity for low-abundance DTX3 detection

  • Each biotinylated antibody can bind multiple streptavidin molecules, each carrying multiple detection molecules

• Flexibility in detection modality:

  • The same biotin-conjugated antibody can be used with various streptavidin conjugates (fluorescent dyes, enzymes, quantum dots)

  • Facilitates adaptation to different imaging platforms without requiring multiple directly-labeled antibodies

• Extended shelf-life:

  • Biotin conjugates typically show greater stability than direct fluorophore conjugates

  • Less susceptible to photobleaching during storage

• Compatibility with pretargeting strategies:

  • Enables sophisticated multi-step imaging protocols like the three-step pretargeting approach

  • Allows for improved target-to-background ratios in specialized applications

Limitations:

• Endogenous biotin interference:

  • Tissues containing high levels of endogenous biotin (liver, kidney, brain) may show background staining

  • Requires additional blocking steps not needed with directly labeled antibodies

• Multi-step protocols:

  • Additional incubation and washing steps increase protocol complexity and duration

  • Each step introduces potential variability in staining quality

• Steric hindrance in multiplex applications:

  • The larger size of streptavidin-based detection systems can limit epitope accessibility in densely labeled samples

  • May complicate co-localization studies with closely adjacent proteins

• Cross-reactivity concerns:

  • Streptavidin can bind non-specifically to certain tissue components

  • May require more extensive blocking and optimization than direct conjugates

• Resolution limitations in super-resolution microscopy:

  • The additional size of the biotin-streptavidin complex (compared to direct labeling) can limit spatial resolution in techniques like STORM or PALM

For optimal application selection, researchers should consider the abundance of DTX3 in their sample, required detection sensitivity, and the specific imaging modalities being employed. For routine detection of abundant DTX3, direct conjugates may offer simplicity, while biotin conjugates excel when maximum sensitivity and system flexibility are required.

How can I design experiments to investigate DTX3's E3 ubiquitin ligase activity using biotin-conjugated antibodies in conjunction with proteomics approaches?

Designing experiments to investigate DTX3's E3 ubiquitin ligase activity requires integrating biotin-conjugated antibodies with sophisticated proteomics techniques. This methodological framework enables comprehensive characterization of DTX3's enzymatic function:

• Substrate identification using BioID proximity labeling:

  • Generate DTX3-BirA* fusion protein expression constructs

  • BirA* will biotinylate proteins in close proximity to DTX3

  • Isolate biotinylated proteins using streptavidin pulldown

  • Identify potential substrates via mass spectrometry

  • Validate candidates using biotin-conjugated DTX3 antibodies in co-immunoprecipitation

• Ubiquitination site mapping:

  • Immunoprecipitate DTX3 and its substrates using biotin-conjugated DTX3 antibody

  • Perform in vitro ubiquitination assays with identified substrates

  • Digest proteins and enrich for ubiquitinated peptides (using di-Gly remnant antibodies)

  • Identify ubiquitination sites via LC-MS/MS analysis

  • Compare ubiquitination patterns with and without active DTX3

• DTX3 interactome analysis:

  • Use biotin-conjugated DTX3 antibody for immunoprecipitation under native conditions

  • Identify interacting proteins via mass spectrometry

  • Focus analysis on ubiquitination machinery components (E2 enzymes, substrate adaptors)

  • Validate interactions using reciprocal immunoprecipitation and Western blotting

• Ubiquitin chain topology analysis:

  • Immunoprecipitate DTX3 substrates after cellular expression of tagged ubiquitin mutants

  • Use biotin-conjugated DTX3 antibody to confirm substrate identity

  • Determine chain types (K48, K63, etc.) using chain-specific antibodies

  • Correlate chain topology with substrate fate (degradation vs. signaling)

• In vivo ubiquitination dynamics:

  • Establish cellular systems with manipulated DTX3 levels (overexpression, knockdown)

  • Use biotin-conjugated DTX3 antibody to monitor DTX3 localization and expression

  • Quantify substrate levels and ubiquitination status in response to Notch pathway stimulation

  • Employ cycloheximide chase experiments to assess substrate stability

This integrated approach leverages the specificity of biotin-conjugated DTX3 antibodies with the analytical power of modern proteomics to comprehensively characterize DTX3's E3 ligase activity, substrate preference, and regulatory mechanisms within the Notch signaling pathway context .