DTX6 Antibody

What is DR6 and why is it relevant to antibody research?

DR6 (Death receptor 6), also known as TNFRSF21, is a death domain-containing receptor belonging to the TNFR superfamily. It is widely expressed in human tissues and cell lines, playing roles in apoptosis induction and activation of signaling pathways including NF-kappaB and JNK . DR6 is particularly interesting because its natural ligand remains undefined, making antibodies crucial tools for studying its function . Research with DR6-deficient mice indicates it functions as a regulatory receptor for CD4+ T cell activation and maintains proper immune responses .

What are the structural features of DR6 that antibodies can target?

DR6 contains multiple cysteine-rich domains (CRDs) in its extracellular portion, with functional agonist antibodies like DQM3 binding specifically to the first CRD . The protein also contains an intracellular death domain (DD) essential for its signaling function. The recombinant human DR6 used as an immunogen for producing the Luke-1 monoclonal antibody comprised amino acids 42-335 , suggesting this region contains important epitopes for antibody recognition.

What types of DR6 antibodies are currently available for research?

From the search results, two main types of DR6 antibodies are identified:

• Functional agonist monoclonal antibodies (e.g., DQM3) that bind to the first cysteine-rich domain and activate DR6 signaling

• Detection antibodies (e.g., Luke-1) that recognize human DR6 and can be used in techniques such as flow cytometry

How do agonist antibodies activate DR6 downstream signaling pathways?

The agonist monoclonal antibody DQM3 activates DR6 signaling through binding to the first cysteine-rich domain. This activation is dependent on DR6's intracellular death domain. Research demonstrates that DQM3 stimulation enhances the association between DR6 and TRADD (TNFR-associated death domain protein), and TRADD is directly involved in DR6-induced signaling activation . This mechanism provides important insights into how DR6 transmits signals following activation, even in the absence of its natural ligand.

What methodologies are most effective for studying DR6-antibody interactions?

Based on the search results, several approaches have proven effective:

• Flow cytometry: For detecting DR6 expression and antibody binding on cell surfaces

• Protein interaction analysis: To study associations between DR6 and adapter proteins like TRADD following antibody stimulation

• Signaling pathway analysis: Investigating JNK activation and other downstream pathways after DR6 stimulation

• Genetic approaches: Using siRNA to knockdown DR6 or associated proteins to confirm specificity of observed effects

For flow cytometry specifically, researchers should prepare approximately 5×10^5 cells, incubate with anti-DR6 antibody (1μg/ml) in FACS buffer (PBS, 5% FCS, 0.02% azide) on ice for 30 minutes, followed by appropriate secondary antibody detection .

How can computational approaches improve DR6 antibody design?

While not specifically addressing DR6 antibody design, current computational methods for antibody development offer relevant insights. The AbDesign algorithm operates in three critical stages:

• Segmentation and recombination of natural antibody Fv backbones

• Docking newly designed backbones against target antigenic surfaces

• Sampling different conformations and optimizing sequences using Rosetta design calculations

Two essential criteria for successful antibody design include preserving amino acid identities crucial for configuring the Fv backbone (including buried polar networks) and adopting proper segmentation to retain intricate hydrogen bonding patterns observed in natural antibodies . These principles could guide development of improved DR6-targeting antibodies.

What are optimal conditions for storage and handling of DR6 antibodies?

For maintaining DR6 antibody integrity:

• Store long-term at -20°C

• Store short-term at 4°C

• Avoid freeze/thaw cycles

• When stored as recommended, antibodies remain stable for at least 1 year

If small volumes become entrapped in the vial's seal during shipment, briefly centrifuge to dislodge any liquid in the container's cap .

What controls are essential when using DR6 antibodies in experimental procedures?

Critical controls include:

• Negative controls: For flow cytometry, use mock-transfected cells to establish baseline fluorescence (as shown in search result where HEK 293T cells were mock transfected)

• Specificity controls: Confirm that observed effects are dependent on the intracellular death domain of DR6

• Positive controls: Include samples known to express DR6 when possible

• Secondary antibody controls: Particularly important in multi-color flow cytometry experiments

How can researchers validate DR6 antibody specificity in their experimental systems?

Validation strategies should include:

• Expression testing: Verify DR6 expression in experimental systems using complementary methods

• Functional validation: For agonist antibodies, confirm activation of known DR6 pathways, such as JNK activation or TRADD association

• Competitive binding: Test if unlabeled antibody competes with labeled antibody for binding

• Cross-reactivity testing: Ensure the antibody doesn't recognize related receptors in the TNFR family

How does DR6 signaling relate to other death receptor pathways?

• Its ligand remains unidentified, unlike TNF and Fas ligand for their respective receptors

• Overexpression of DR6 induces apoptosis and activates NF-kappaB and JNK

• DR6 interacts with the death domain-containing adapter molecule TRADD, similar to TNF-R1

These distinctions make DR6 antibodies particularly valuable for comparative studies of death receptor signaling mechanisms.

How can researchers design experiments to investigate DR6's role in T cell biology?

Studies with DR6-deficient mice revealed that DR6 attenuates Th2 differentiation and functions as a regulatory receptor for CD4+ T cell activation . To investigate these functions:

• Compare T cell differentiation in presence of agonist DR6 antibodies versus control antibodies

• Analyze T cell activation markers following DR6 stimulation in different T cell subsets

• Measure cytokine production profiles to assess how DR6 signaling shifts T helper cell polarization

• Use antibody blocking studies to determine if DR6's regulatory functions can be modulated

What methodological approaches are effective for studying DR6-mediated apoptosis?

To investigate DR6's role in programmed cell death:

• Treat cells with agonist antibodies like DQM3 and measure apoptotic markers (caspase activation, phosphatidylserine exposure, DNA fragmentation)

• Compare apoptotic responses in cells with varying DR6 expression levels

• Analyze the kinetics of TRADD recruitment and subsequent signaling events following antibody stimulation

• Use pathway inhibitors to dissect which downstream components are essential for DR6-mediated apoptosis

How might antibody approaches help identify the natural ligand for DR6?

Since DR6's natural ligand remains unidentified , antibody-based approaches could help in discovery efforts:

• Development of antibody arrays to screen for proteins that compete with DR6 antibodies for binding

• Use of DR6-Fc fusion proteins as bait, followed by antibody-based detection of captured ligands

• Antibody inhibition studies in biological systems where DR6 function has been established to identify contexts where ligand-receptor interactions might occur

What are promising applications for DR6 antibodies in immunological research?

Based on DR6's roles in T cell regulation and immune response modulation , DR6 antibodies could be valuable for:

• Investigating mechanisms of CD4+ T cell activation and regulation

• Studying the balance between Th1 and Th2 immune responses

• Exploring DR6's potential role in autoimmune conditions or inflammatory disorders

• Developing immunomodulatory approaches that target DR6 signaling pathways