FADD Antibody
Description
Structure and Function of FADD
FADD contains two key domains:
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Death Domain (DD): Binds to death receptors (e.g., Fas, TNFR1) via electrostatic interactions.
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Death Effector Domain (DED): Recruits procaspases (e.g., caspase-8) to form the death-inducing signaling complex (DISC) .
FADD’s primary role is mediating extrinsic apoptosis, but it also regulates cell proliferation, immune responses, and inflammation. Post-translational modifications, such as phosphorylation at Ser194, modulate its activity in cell cycle arrest and apoptosis .
Applications of FADD Antibody
FADD Antibody is employed in diverse experimental workflows:
Product Specs
Q&A
What is FADD and why is it important in cancer research?
FADD is a 23 kDa adaptor protein officially named "Fas (TNFRSF6)-associated via death domain" that plays a crucial role in death receptor-mediated apoptosis. The protein is widely expressed in various tissues and has been demonstrated to correlate with tumor progression and prognosis . The interaction between FAS and FADD death domains is essential for forming the death-inducing signaling complex (DISC) .
Research has shown FADD expression in multiple cell lines including HT-1080, A549, HeLa, HepG2, and Jurkat cells, as well as in various tissues . Its critical role in cell death pathways makes it a significant target for cancer research, particularly in understanding treatment resistance mechanisms.
What applications are FADD antibodies validated for?
FADD antibodies have been validated for multiple research applications:
| Application | Recommended Dilution | Validated Cell/Tissue Types |
|---|---|---|
| Western Blot (WB) | 1:2000-1:10000 | HT-1080, A549, HeLa, HepG2, Jurkat cells, mouse pancreas tissue |
| Immunohistochemistry (IHC) | 1:500-1:2000 | Human lung cancer tissue, human colon tissue, human cervical cancer tissue, rat/mouse kidney tissue |
| Immunofluorescence (IF)/ICC | 1:200-1:800 | HeLa cells |
| Immunoprecipitation (IP) | 0.5-4.0 μg for 1.0-3.0 mg protein lysate | A549 cells |
| Co-Immunoprecipitation (CoIP) | Varies by protocol | Various |
| ELISA | Application-dependent | Recombinant and native human FADD |
The specific application should be selected based on research requirements. For example, WB is useful for protein expression quantification, while IHC provides spatial information in tissue sections .
What are the key differences between polyclonal and monoclonal FADD antibodies?
For critical quantitative applications where reproducibility is paramount, recombinant monoclonal antibodies offer "unrivalled batch-to-batch consistency, easy scale-up, and future security of supply" .
How should I store and handle FADD antibodies to maintain activity?
Proper storage and handling are critical for maintaining antibody activity:
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Storage temperature: Most FADD antibodies should be stored at -20°C (e.g., 14906-1-AP) or -80°C (e.g., 84619-1-PBS)
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Buffer composition: Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3
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Stability: Generally stable for one year after shipment when stored properly
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Aliquoting: For antibodies stored at -20°C, aliquoting may be unnecessary , but is recommended for frequent use to avoid freeze-thaw cycles
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Working dilutions: Should be prepared fresh and used within 24 hours
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BSA/azide-free options: Available for conjugation applications (e.g., 84619-1-PBS)
Always check the manufacturer's specific recommendations as storage conditions can vary between products.
How can I optimize FADD antibody dilutions for different experimental systems?
Optimization is essential for obtaining reliable results with FADD antibodies. The search results provide recommended dilution ranges, but these should be further optimized for each experimental system:
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Perform titration experiments: Test a range of dilutions around the manufacturer's recommendation (e.g., 1:1000, 1:2000, 1:5000, 1:10000 for WB)
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Consider sample type variation: Different cell lines may require different antibody concentrations. For example, while 1:2000-1:10000 is recommended for WB generally , specific cell lines may require adjustment
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Account for detection method: Chemiluminescence detection systems typically require lower antibody concentrations than colorimetric methods
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Positive controls: Include known FADD-expressing samples (e.g., HeLa cells) to validate detection at each dilution
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Background minimization: If high background is observed, increase dilution or optimize blocking conditions
As stated in the product information: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .
What are the considerations for detecting phosphorylated FADD?
FADD undergoes phosphorylation at multiple sites, with S194 being particularly important for its function. When detecting phosphorylated FADD:
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Phospho-specific antibodies: Use antibodies specifically targeting phosphorylated epitopes, such as the Human Phospho-FADD (S194) Antibody
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Cell cycle considerations: Phosphorylation of FADD at S194 changes during cell cycle. Treatments with nocodazole or hydroxyurea for 20 hours can enhance phospho-FADD detection
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Subcellular localization: Phospho-FADD (S194) has been shown to localize to nuclei in HeLa cells , which differs from total FADD distribution
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Sample preparation: Phosphatase inhibitors must be included in lysis buffers to preserve phosphorylation status
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Detection methods: For immunofluorescence detection of phospho-FADD, protocols using specific conditions (e.g., 10 μg/mL antibody for 3 hours at room temperature) have been validated
Experimental data has shown that "FADD phosphorylated at S194 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line stimulated with nocadazole" , demonstrating the importance of proper cell treatment for phospho-specific detection.
How can I develop a sandwich ELISA for FADD detection?
Developing a sandwich ELISA requires careful selection of paired antibodies:
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Antibody pair selection: Choose antibodies recognizing different epitopes. For example, mAb 3F9 as capture antibody and biotin-conjugated 3A3 as detection antibody have been validated
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Sensitivity determination: Established ELISAs have demonstrated detection limits of 0.3 ng of purified His₆-FADD
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Specificity validation: Confirm specific detection through blocking experiments, such as using rabbit anti-FADD sera to block positive reactions
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Commercial options: Consider matched antibody pairs like MP01426-2 (84619-1-PBS capture and 84619-2-PBS detection) that have been validated in cytometric bead array
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Recombinant antibodies: For consistent results, recombinant antibody production enables "unrivalled batch-to-batch consistency, easy scale-up, and future security of supply"
The advantage of ELISA over traditional Western blot or immunohistochemistry approaches is higher throughput and potentially greater quantitative precision .
What controls should be included when using FADD antibodies?
Proper experimental controls are essential for reliable FADD detection:
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Positive controls: Include known FADD-expressing samples such as:
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Negative controls:
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Primary antibody omission control
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Non-specific IgG controls matching the host species of the primary antibody
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Cell lines with FADD knockdown or knockout (if available)
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Treatment controls: For phospho-FADD detection, include both treated (+) and untreated (-) samples (e.g., with nocodazole or hydroxyurea)
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Loading controls: For Western blot, include housekeeping proteins detection
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Antibody validation: Consider blocking experiments with competing peptides or using rabbit anti-FADD sera to confirm specificity
What are common troubleshooting strategies for FADD antibody experiments?
For antigen retrieval in IHC applications, data indicates that "suggested antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" .
How can I use FADD antibodies in multiplex immunofluorescence studies?
For multiplex detection combining FADD with other proteins:
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Antibody selection: Choose antibodies from different host species or different isotypes to avoid cross-reactivity
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Conjugated antibodies: Consider using directly conjugated antibodies or sequential detection protocols
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Validated combinations: For phospho-FADD (S194), successful detection has been demonstrated using:
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Conjugation-ready formats: Some FADD antibodies are available in BSA and azide-free storage buffer at 1 mg/mL, specifically designed for conjugation applications
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Validated applications: "This conjugation ready format makes antibodies ideal for use in many applications including: ELISAs, multiplex assays requiring matched pairs, mass cytometry, and multiplex imaging applications"
What are the post-translational modifications of FADD and how do they affect detection?
FADD undergoes several post-translational modifications that can affect its detection and function:
The observed molecular weight of FADD ranges from 23-30 kDa , which likely reflects these post-translational modifications. When selecting antibodies, consider whether the epitope includes or is near sites of post-translational modification.
How do different fixation and antigen retrieval methods affect FADD detection in tissues?
Fixation and antigen retrieval significantly impact FADD detection in IHC applications:
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Recommended antigen retrieval:
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Fixation considerations:
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Tissue-specific optimization:
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Controls for optimization:
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Include positive control tissues with known FADD expression
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Test multiple retrieval conditions on the same tissue type
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Optimization of these parameters is essential as "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .
How can FADD antibodies be used to study apoptotic signaling pathways?
FADD antibodies can provide insights into apoptotic signaling through various approaches:
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Co-immunoprecipitation (CoIP): Detect FADD interactions with death receptors and other components of the DISC complex
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Phosphorylation status: Monitor changes in FADD phosphorylation (particularly at S194) in response to treatments or during cell cycle progression
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Subcellular localization: Track FADD redistribution during apoptosis induction using immunofluorescence (IF/ICC approaches at 1:200-1:800 dilution)
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Expression correlation: Correlate FADD expression levels with sensitivity to apoptotic stimuli across different cell types
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Protein complex formation: Use FADD antibodies in combination with other apoptotic pathway proteins to monitor DISC formation
The translocation and post-translational modifications of FADD are critical for understanding its role in death receptor signaling and cell cycle regulation.
What are the considerations when comparing FADD expression across different sample types?
When comparing FADD expression across different samples:
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Different cell types: FADD expression has been confirmed in multiple cell lines including HT-1080, A549, HeLa, HepG2, and Jurkat cells , but quantitative differences exist
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Tissue versus cell lines: Expression patterns may differ between cultured cells and tissue samples
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Normalization strategies:
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For Western blot: Normalize to appropriate loading controls
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For IHC: Consider using tissue microarrays with internal controls
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Detection methods: Different methods (WB, IHC, ELISA) may yield different quantitative results and should not be directly compared
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Antibody selection: Using the same antibody clone across all samples is crucial for comparative studies
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Species considerations: While FADD is conserved, species-specific differences exist. Confirm antibody reactivity (human, mouse, rat reactivity has been validated for many antibodies)
How should I interpret variations in FADD molecular weight across different samples?
The calculated molecular weight of FADD is 23 kDa, but observed molecular weights range from 23-30 kDa . This variation can provide important functional information:
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Post-translational modifications: Phosphorylation and sumoylation can increase apparent molecular weight
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Cell-type specificity: Different cell types may exhibit different patterns of FADD modification
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Treatment effects: Treatments that affect cell cycle (e.g., nocodazole or hydroxyurea) can alter phosphorylation status and molecular weight
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Isoform expression: Consider potential alternative splicing or proteolytic processing
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Technical considerations:
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Gel percentage affects migration and apparent molecular weight
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Different buffer systems may result in slight variations in migration patterns
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When reporting molecular weight variations, always include the experimental context and detection method.
What advanced applications utilize FADD antibodies beyond standard protein detection?
Beyond standard detection methods, FADD antibodies enable sophisticated research applications:
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Proximity ligation assays: Detect protein-protein interactions between FADD and binding partners with spatial resolution
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Chromatin immunoprecipitation (ChIP): For studies examining potential nuclear functions of FADD
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Flow cytometry: Quantify FADD expression levels in heterogeneous cell populations
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Mass cytometry: Combine FADD detection with numerous other markers using conjugation-ready antibody formats
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In vivo imaging: Using appropriately labeled FADD antibodies in animal models
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Cytometric bead arrays: Using matched antibody pairs like MP01426-2 (84619-1-PBS capture and 84619-2-PBS detection)
These advanced applications require careful antibody validation and optimization but provide powerful insights into FADD biology beyond what standard methods can reveal.
What emerging technologies are improving FADD detection and functional studies?
Emerging technologies are enhancing our ability to study FADD:
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Recombinant antibody production: Provides "unrivalled batch-to-batch consistency, easy scale-up, and future security of supply"
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Single-cell proteomics: Allowing examination of FADD expression and modification at the single-cell level
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CRISPR-engineered cellular models: Creating precise FADD mutations or tagging endogenous FADD for functional studies
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Live-cell imaging probes: Developing tools to visualize FADD dynamics in living cells
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Multiplex detection platforms: Enabling simultaneous analysis of FADD with multiple other proteins in complex signaling networks
As research continues, these technologies will provide increasingly detailed insights into FADD's roles in apoptosis, cell cycle regulation, and disease processes.
Product Science Overview
The Fas-Associated Death Domain (FADD) is a crucial adaptor protein involved in the transduction of apoptotic signals from death receptors (DRs) to initiate programmed cell death. This protein plays a significant role in maintaining cellular homeostasis by eliminating damaged, infected, or unwanted cells. FADD is also known as MORT1 and is encoded by the FADD gene located on chromosome 11 in humans . The mouse anti-human FADD antibody is commonly used in research to study the protein’s function and its role in various cellular processes.
FADD is a 23 kDa protein composed of 208 amino acids. It contains two main domains: the C-terminal death domain (DD) and the N-terminal death effector domain (DED). These domains, although structurally similar, have distinct functions. The DD of FADD binds to the DD of death receptors such as the Fas receptor, while the DED interacts with the DED of intracellular molecules like procaspase-8 .
Upon ligand binding to the Fas receptor, the receptor trimerizes, and FADD binds to the DD of this trimeric structure. This interaction unmask FADD’s DED, allowing it to recruit procaspase-8 and -10, forming the death-inducing signaling complex (DISC). The activation of these procaspases initiates the caspase cascade, leading to apoptosis .
FADD is predominantly known for its role in apoptosis, but it also participates in other cellular processes such as cell proliferation, autophagy, necroptosis, and inflammation. The protein’s involvement in these processes highlights its versatility and importance in regulating cell survival and proliferation .
- Apoptosis: FADD is essential for the extrinsic pathway of apoptosis, where it mediates the formation of the DISC and the subsequent activation of initiator caspases .
- Autophagy: FADD has been implicated in autophagy, a process that degrades and recycles cellular components to maintain cellular homeostasis .
- Necroptosis: FADD is involved in necroptosis, a form of programmed necrosis that serves as an alternative cell death pathway when apoptosis is inhibited .
- Inflammation: FADD plays a role in inflammatory signaling, contributing to immune responses and cellular homeostasis .
The mouse anti-human FADD antibody is widely used in research to study the protein’s function and its role in various cellular processes. This antibody is particularly useful in immunohistochemistry, Western blotting, and flow cytometry to detect and quantify FADD expression in different cell types and tissues.
