DLL1 Antibody

What is DLL1 and what cell types express it?

DLL1 is a type I transmembrane protein in the Delta/Serrate/Lag-2 (DSL) family of Notch ligands. Mature rat DLL1 consists of a 520 amino acid extracellular domain (ECD) with one DSL domain and eight EGF-like repeats, a 23 amino acid transmembrane segment, and a 154 amino acid cytoplasmic domain . DLL1 is primarily expressed by thymic and splenic stromal cells, macrophages, and dendritic cells . During development, it is prominently expressed in embryonic tissues such as the mouse stomach at E13.5 . In pathological conditions, DLL1 expression has been detected in various cancer cells, including breast cancer tissue, HepG2 human hepatocellular carcinoma cells, and other cell lines like MG-63, HeLa, and HUVEC .

How do different DLL1 antibodies compare in terms of species reactivity?

Based on current research, several species-specific DLL1 antibodies are available for experimental applications:

When selecting an antibody, it's critical to match the species reactivity to your experimental model. For cross-species studies, antibodies like 28544-1-AP offer broader reactivity across human, mouse, and rat samples .

What is the molecular weight of DLL1 detected in Western blots?

The calculated molecular weight of DLL1 is approximately 78 kDa, but the observed molecular weight in Western blots varies between 70-100 kDa depending on post-translational modifications and proteolytic processing . The 28544-1-AP antibody typically detects DLL1 at approximately 70 kDa in rat brain tissue, MG-63 cells, HeLa cells, HUVEC cells, and mouse brain tissue . These variations in observed molecular weight are attributed to glycosylation patterns and ADAM9, 12, or 17-mediated proteolysis, which releases a 60 kDa ECD fragment and affects DLL1 function in regulating Notch-dependent processes .

Do DLL1 antibodies cross-react with other Delta-like proteins?

Most commercial DLL1 antibodies are designed for high specificity. For example, the Human DLL1 Antibody (MAB18181) shows no cross-reactivity with recombinant human DLL3 or DLL4 in direct ELISAs . Similarly, rat DLL1 shares only 26%, 36%, and 53% amino acid sequence identity with rat DLL2, DLL3, and DLL4, respectively, which helps in developing specific antibodies . When selecting an antibody for experiments where specificity is crucial, researchers should carefully review cross-reactivity data provided by manufacturers and consider performing their own validation experiments.

What are the recommended applications and dilutions for DLL1 antibodies?

Different DLL1 antibodies are optimized for specific applications. Based on current research data:

For optimal results in each application, researchers should perform titration experiments with their specific samples and experimental conditions to determine the ideal antibody concentration .

How should I optimize immunostaining protocols for DLL1 detection?

For successful DLL1 immunostaining, consider these methodological approaches:

For paraffin-embedded tissue sections:

• Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic

• Incubate with primary antibody (e.g., MAB18181 at 1.7 μg/mL) for 1 hour at room temperature

• Detect using an appropriate secondary antibody system (e.g., Anti-Mouse IgG HRP Polymer)

• Develop with DAB and counterstain with hematoxylin

For frozen tissue sections:

• Fix sections appropriately (e.g., PFA fixation)

• Apply primary antibody (e.g., AF3970 at 10 μg/mL) overnight at 4°C

• Visualize using fluorophore-conjugated secondary antibodies (e.g., NorthernLights 557-conjugated Anti-Sheep IgG)

• Counterstain with DAPI for nuclear visualization

For cultured cells:

• Fix cells (typically with PFA)

• Apply primary antibody (e.g., MAB18181 at 25 μg/mL) for 3 hours at room temperature

• Detect using fluorescently-labeled secondary antibody

• Counterstain nuclei with DAPI

What positive controls should I include when validating DLL1 antibody performance?

Based on published research, recommended positive controls include:

• For mouse/rat DLL1 antibodies: Mouse splenocytes, embryonic mouse stomach (E13-E13.5), mouse brain tissue

• For human DLL1 antibodies: HepG2 cells, human breast cancer tissue, MG-63 cells, HeLa cells, HUVEC cells

• Engineered controls: DLL1-transfected CHO cells

When designing validation experiments, including both positive and negative controls is essential. For negative controls, consider using isotype control antibodies (e.g., polyclonal Armenian hamster IgG for HMD1-5 ) and tissues/cells known to lack DLL1 expression.

How can I improve DLL1 detection in flow cytometry experiments?

For optimal DLL1 detection in flow cytometry:

• Cell preparation: Ensure single-cell suspensions with minimal cell death

• Antibody titration: Determine optimal antibody concentration using positive control samples (e.g., mouse splenocytes for AF3970)

• Proper controls: Include isotype control antibodies to assess non-specific binding

• Secondary antibody selection: Use appropriate fluorochrome-conjugated secondary antibodies (e.g., NorthernLights 637-conjugated Anti-Sheep IgG for AF3970)

• Protocol optimization: Follow manufacturer's protocols for staining membrane-associated proteins

• Instrument settings: Establish appropriate compensation settings if using multiple fluorochromes

The detection of DLL1 in mouse splenocytes using AF3970 has been successfully demonstrated, suggesting these cells as an appropriate positive control for flow cytometry experiments .

How can neutralizing DLL1 antibodies be used to study Notch signaling pathways?

Neutralizing DLL1 antibodies provide powerful tools for dissecting the specific contributions of DLL1-Notch signaling in various biological processes:

What is known about the role of DLL1 in breast cancer and how can antibodies help study this?

DLL1 has emerged as a significant player in breast cancer biology, particularly in ER+ breast cancer:

• Cancer cell properties: DLL1 promotes cancer cell colony formation, proliferation, survival, migration, and invasion in breast cancer models .

• Cancer stem cell function: DLL1+ cells display similarities to cancer stem cells, exhibiting high tumor-initiating capacity and the ability to drive metastasis formation .

• Therapeutic resistance: DLL1+ cells contribute to chemoresistance in aggressive luminal breast tumors, highlighting DLL1 as a potential therapeutic target .

The novel antibody Dl1.72 demonstrates the potential of anti-DLL1 approaches:

• Binds to human DLL1 with nanomolar affinity

• Impairs DLL1-Notch signaling in ER+ breast cancer cells

• Reduces cancer cell proliferation, migration, and mammosphere formation

• Inhibits endothelial tube formation (suggesting anti-angiogenic effects)

• In xenograft models, significantly inhibits tumor growth and liver metastasis without apparent toxicity

These findings suggest that anti-DLL1 antibodies could serve as valuable tools for studying the complex roles of DLL1 in breast cancer and potentially as therapeutic agents, particularly in cases of endocrine resistance .

How can DLL1 antibodies be used to investigate cellular localization and trafficking?

The subcellular localization of DLL1 provides important insights into its function and regulation. Researchers can use DLL1 antibodies to:

• Study membrane localization: DLL1 functions primarily at the cell membrane where it interacts with Notch receptors on adjacent cells. Immunofluorescence studies have shown co-localization of DLL1 with Nectin-2 at the cell membrane, which can be disrupted under certain conditions .

• Investigate proteolytic processing: DLL1 undergoes ADAM9, 12, or 17-mediated proteolysis, releasing a 60 kDa ECD fragment, followed by presenilin-dependent cleavage of the membrane-bound portion. Antibodies recognizing different domains can track these processing events .

• Examine cell density effects: Studies have shown differences in DLL1 localization between sparse and confluent cell cultures, suggesting regulation by cell-cell contact. Antibodies allow visualization of these changes .

• Analyze co-localization with interaction partners: DLL1 antibodies can be used alongside antibodies against other proteins (like Nectin-2) to study spatial relationships at the subcellular level through confocal microscopy .

Why might I observe multiple bands in Western blot when using DLL1 antibodies?

Multiple bands in DLL1 Western blots may reflect several biological and technical factors:

• Proteolytic processing: DLL1 undergoes sequential proteolytic processing by ADAM proteases and presenilin-dependent enzymes, generating fragments of different molecular weights .

• Glycosylation heterogeneity: As a transmembrane protein, DLL1 undergoes post-translational modifications, particularly glycosylation, which can result in heterogeneous migration patterns.

• Splice variants: Alternative splicing may generate different DLL1 isoforms that are recognized by the same antibody.

• Sample preparation effects: Insufficient denaturation, partial degradation during sample preparation, or incomplete reduction of disulfide bonds can all contribute to multiple banding patterns.

To address this issue:

• Optimize sample preparation conditions (buffer composition, protease inhibitors)

• Consider using antibodies targeting different epitopes to confirm specificity

• Include appropriate positive controls (e.g., recombinant DLL1 protein)

• Perform peptide competition assays to verify band specificity

What factors might lead to inconsistent DLL1 immunostaining patterns?

Inconsistent DLL1 immunostaining may result from several factors:

• Biological variability in expression: DLL1 expression can vary based on cell type, developmental stage, and physiological conditions .

• Epitope masking: The accessibility of DLL1 epitopes may be affected by protein-protein interactions or conformational changes.

• Fixation and processing variables: Different fixation methods can affect epitope preservation. For example, in immunohistochemistry of embryonic mouse stomach, DLL1 detection required specific fixation and antigen retrieval methods .

• Cell culture conditions: DLL1 localization and expression can be affected by cell culture density, with differences observed between sparse and confluent cultures .

• Antibody clone differences: Different antibody clones recognize distinct epitopes on DLL1, potentially leading to different staining patterns.

To improve consistency:

• Standardize fixation and processing protocols

• Optimize antigen retrieval conditions (temperature, pH, duration)

• Include positive and negative controls in each experiment

• Use consistent cell culture conditions

• Consider using multiple antibodies targeting different DLL1 epitopes

How can I validate the specificity of DLL1 antibody signals in my experiments?

Comprehensive validation of DLL1 antibody specificity should include:

• Multiple detection methods: Compare results across different techniques (e.g., Western blot, IHC, flow cytometry) using the same antibody.

• Positive and negative controls: Include known DLL1-expressing samples (e.g., embryonic mouse stomach, transfected CHO cells, HepG2 cells) and negative controls (tissues lacking DLL1 expression) .

• Isotype controls: Use appropriate isotype control antibodies (e.g., polyclonal Armenian hamster IgG for HMD1-5) to assess non-specific binding .

• Peptide competition assays: Pre-incubate the antibody with recombinant DLL1 protein before staining to confirm signal specificity.

• siRNA/shRNA knockdown: Demonstrate reduction in antibody signal following DLL1 knockdown.

• Genetic models: When available, use DLL1 knockout/knockdown tissues or cells as negative controls.

• Cross-validation with multiple antibodies: Use antibodies from different sources or targeting different epitopes to confirm staining patterns.

How are therapeutic anti-DLL1 antibodies being developed for cancer treatment?

The development of therapeutic anti-DLL1 antibodies represents an important frontier in targeted cancer therapy:

• Antibody development strategies: The novel anti-DLL1 antibody Dl1.72 was developed using phage display technology, selecting an anti-DLL1 antibody fragment which was subsequently converted into a full human IgG1. This approach yielded an antibody with nanomolar affinity for human DLL1 and no binding to other Notch ligands .

• Preclinical validation: Rigorous testing has demonstrated that Dl1.72:

  • Impairs DLL1-Notch signaling and reduces Notch target gene expression in ER+ breast cancer cells

  • Reduces cancer cell proliferation, migration, and mammosphere formation in vitro

  • Inhibits tumor growth and liver metastasis in xenograft models without apparent toxicity

• Comparative advantages: Antibody-targeting of individual Notch components like DLL1 is expected to have superior therapeutic efficacy compared to complete Notch inhibitors, with better tolerability and reduced side effects .

• Production considerations: For therapeutic applications, antibodies must be produced under specific conditions. For example, Dl1.72 was produced in HEK293E6 suspension cells and purified in endotoxin-free conditions to ensure safety for in vivo applications .

These developments suggest that anti-DLL1 antibodies hold promise as cancer therapeutics, either as standalone treatments or in combination with conventional therapies, particularly for endocrine-resistant ER+ breast cancer .

How can DLL1 antibodies contribute to our understanding of cancer stem cells?

DLL1 antibodies are providing valuable insights into cancer stem cell (CSC) biology:

• CSC identification and characterization: DLL1+ cells have been shown to bear similarities to CSCs, exhibiting high tumor-initiating capacity. Antibodies enable the identification and isolation of these cells for further study .

• Functional studies: Neutralizing antibodies like Dl1.72 reduced mammosphere formation in vitro, suggesting that DLL1 signaling supports the cancer stem cell population. This provides a tool to study the functional requirements of DLL1 in maintaining stemness .

• Therapeutic targeting: The ability of DLL1 antibodies to reduce both primary tumor growth and metastasis formation in breast cancer models suggests that targeting DLL1+ CSCs may be an effective therapeutic strategy .

• Resistance mechanisms: DLL1+ cells contribute to chemoresistance in aggressive luminal breast tumors. Antibodies that can identify and potentially target these resistant populations offer new approaches to overcome therapy resistance .

• Lineage tracing: In developmental and cancer studies, antibodies against DLL1 can help track the fate of DLL1-expressing progenitor cells, providing insights into lineage relationships and hierarchical organization within tumors.

What future research directions are emerging for DLL1 antibodies?

Several promising research directions are emerging for DLL1 antibodies:

• Combination therapies: DLL1-targeting therapies are expected to provide clinical benefits when used alone and in combination with conventional chemotherapy or endocrine therapy, particularly for ER+ breast cancer .

• Expanded cancer applications: While current research focuses on ER+ breast cancer, the role of DLL1 in other cancer types warrants investigation, potentially expanding the therapeutic applications of DLL1 antibodies.

• Enhanced antibody engineering: Development of antibody derivatives with improved properties, such as antibody-drug conjugates (ADCs), bispecific antibodies, or antibodies with modified Fc regions for enhanced effector functions.

• Biomarker development: Research into DLL1 expression as a biomarker for patient stratification and prediction of response to various therapies represents an important direction.

• Developmental biology applications: Beyond cancer research, DLL1 antibodies continue to be valuable tools for studying the role of Notch signaling in normal development and tissue homeostasis, particularly in stem cell biology.

• Imaging applications: Development of labeled DLL1 antibodies for in vivo imaging could provide non-invasive methods to monitor DLL1 expression in developmental processes and disease progression.