FADD (Ab-191) Antibody

What is FADD (Ab-191) Antibody and what epitope does it recognize?

FADD (Ab-191) Antibody is a rabbit polyclonal antibody that specifically recognizes the C-terminal region of the Fas-associated death domain (FADD) protein. This antibody targets the region surrounding amino acid 191, which is a critical region for FADD function in apoptotic signaling pathways. The immunogen used for generating this antibody is a synthesized peptide derived from the C-terminal region of human FADD . Unlike phospho-specific antibodies that recognize only phosphorylated forms, the standard FADD (Ab-191) antibody detects total FADD protein regardless of phosphorylation status.

What are the validated applications for FADD (Ab-191) Antibody?

The FADD (Ab-191) Antibody has been validated for multiple experimental applications, primarily:

• Western Blotting (WB): Recommended dilutions of 1:500-1:3000

• Enzyme-Linked Immunosorbent Assay (ELISA): Recommended dilutions of 1:2000-1:10000

• Immunohistochemistry (IHC): Used less frequently than WB

Each application requires specific optimization parameters for maximum sensitivity and specificity in detecting FADD protein in complex biological samples .

What species reactivity has been confirmed for FADD (Ab-191) Antibody?

The FADD (Ab-191) Antibody has confirmed reactivity against both human and mouse FADD protein . The high sequence homology between species in the C-terminal region allows this cross-reactivity. Researchers should note that while the antibody targets human FADD, experimental validation has demonstrated strong mouse reactivity, making it suitable for comparative studies across these species. Some researchers have reported potential rat reactivity, though this may require additional validation for specific experimental contexts.

How should FADD (Ab-191) Antibody be stored and handled for optimal performance?

For optimal performance and stability:

• Store the antibody at -20°C to -80°C in small aliquots to avoid repeated freeze-thaw cycles

• The antibody is typically supplied in a buffer containing rabbit IgG in phosphate-buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Allow the antibody to equilibrate to room temperature before opening the vial

• Centrifuge briefly before opening to ensure recovery of all contents

• For long-term storage, maintain antibody aliquots at -20°C, as repeated freeze-thaw cycles significantly decrease antibody activity

What are the recommended protocol modifications for detecting FADD in Western blotting experiments?

For optimal Western blotting results with FADD (Ab-191) Antibody:

• Sample preparation:

  • Include protease inhibitors in lysis buffers to prevent degradation

  • For phosphorylation studies, add phosphatase inhibitors

  • Use RIPA or NP-40 based lysis buffers for efficient extraction

• Gel electrophoresis and transfer:

  • 10-12% acrylamide gels provide optimal resolution for the 23-25 kDa FADD protein

  • Transfer to PVDF membranes is preferred over nitrocellulose for stronger protein binding

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Use primary antibody at 1:500-1:2000 dilution for standard applications

  • Incubate overnight at 4°C for maximum sensitivity

  • FADD protein typically appears at approximately 23-25 kDa

• Detection considerations:

  • Enhanced chemiluminescence (ECL) detection systems provide sufficient sensitivity

  • Stripping and reprobing may affect epitope recognition; prepare duplicate blots for multiple targets

What controls should be included when using FADD (Ab-191) Antibody in experimental designs?

Proper experimental controls are essential for accurate interpretation of results:

• Positive controls:

  • Cell lines with known FADD expression (e.g., Jurkat cells)

  • Recombinant FADD protein as a size standard

• Negative controls:

  • FADD knockout or knockdown samples

  • Secondary antibody-only controls to assess non-specific binding

  • Peptide competition assays using the immunizing peptide

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH) for normalization

  • Total protein staining methods (Ponceau S, REVERT)

• Validation controls:

  • Additional anti-FADD antibodies targeting different epitopes

  • Comparison with phospho-specific FADD antibodies when studying phosphorylation states

How can FADD (Ab-191) Antibody be used to investigate the role of FADD in apoptosis signaling pathways?

FADD (Ab-191) Antibody is a valuable tool for investigating apoptosis signaling:

• Death-inducing signaling complex (DISC) analysis:

  • Use for co-immunoprecipitation to identify FADD interaction partners within the DISC

  • Combine with antibodies against caspase-8, Fas receptor, or TRAIL receptors to study DISC formation

  • Observe recruitment dynamics following death receptor stimulation

• Apoptotic pathway monitoring:

  • Track FADD expression levels during apoptosis induction

  • Correlate FADD recruitment with downstream caspase activation

  • Compare with phospho-specific antibodies to examine how phosphorylation affects FADD function

• Experimental approaches:

  • Treat cells with apoptosis inducers (TNF-α, FasL, TRAIL) and monitor FADD recruitment

  • Use time-course experiments to track FADD participation in signaling cascades

  • Apply proteasome inhibitors to prevent degradation of apoptotic proteins

How does FADD (Ab-191) Antibody compare with phospho-specific FADD antibodies in research applications?

The use of FADD (Ab-191) Antibody versus phospho-specific variants provides different insights:

What precautions should be taken when using FADD (Ab-191) Antibody in cancer research studies?

When using FADD (Ab-191) Antibody in cancer research, several considerations are important:

• Expression variability:

  • FADD expression is frequently altered in cancer, with studies showing loss of expression in certain cancers like acute myeloid leukemia (AML)

  • Compare multiple cancer cell lines and normal tissue counterparts

  • Consider potential epitope masking in cancer cells due to protein interactions

• Technical considerations:

  • Optimize extraction methods for different cancer types (solid tumors vs. hematological malignancies)

  • Validate antibody specificity in each experimental cancer model

  • Include appropriate positive controls from cell lines with known FADD expression

• Interpretation challenges:

  • Distinguish between total loss of FADD and relocalization

  • Consider post-translational modifications that might affect epitope recognition

  • Assess potential correlation between FADD levels and treatment resistance

What are common issues encountered with FADD (Ab-191) Antibody in Western blotting and how can they be resolved?

Common troubleshooting scenarios for Western blotting with FADD (Ab-191) Antibody:

• No signal or weak signal:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

  • Extend incubation time (overnight at 4°C)

  • Enhance protein loading (25-50 μg of total protein)

  • Check transfer efficiency with reversible protein stains

  • Verify sample preparation methods to ensure protein integrity

• Multiple bands or non-specific signals:

  • Increase blocking stringency (5% BSA instead of milk)

  • Optimize antibody dilution (test dilution series)

  • Add 0.1% Tween-20 to antibody dilution

  • Perform peptide competition assay to identify specific bands

  • Note that FADD may appear as multiple bands due to post-translational modifications

• Inconsistent results between experiments:

  • Use freshly prepared samples to minimize degradation

  • Standardize lysate preparation protocols

  • Include internal controls for normalization

  • Consider the impact of cell confluency and culture conditions on FADD expression

How can FADD (Ab-191) Antibody be effectively used in studying the non-apoptotic functions of FADD in inflammation and immunity?

FADD has emerging roles in inflammation and immunity that can be studied using FADD (Ab-191) Antibody:

• Inflammasome regulation studies:

  • Examine FADD's role in the priming and activation of the NLRP3 inflammasome

  • Monitor FADD expression during inflammatory stimuli exposure

  • Use with caspase-8 antibodies to investigate their cooperative function in inflammation

• Experimental approaches:

  • Stimulate cells with LPS or other TLR ligands to activate inflammatory pathways

  • Compare FADD recruitment in canonical vs. non-canonical inflammasome pathways

  • Use cell fractionation to track FADD's subcellular localization during inflammatory responses

• Technical considerations:

  • Combine with antibodies against NLRP3, ASC, and caspase-1 to study inflammasome complex formation

  • Use cytokine production (IL-1β, IL-18) as functional readouts of inflammasome activity

  • Employ proximity ligation assays to detect FADD interactions with inflammasome components

How can FADD (Ab-191) Antibody be used in combination with other techniques to study FADD phosphorylation and its impact on cellular localization?

Combining FADD (Ab-191) Antibody with complementary techniques provides deeper insights:

How might new applications of FADD (Ab-191) Antibody advance our understanding of FADD's role in tumor suppression?

Emerging applications for FADD (Ab-191) Antibody in cancer research:

• Prognostic biomarker development:

  • FADD expression loss has been associated with poor prognosis in acute myeloid leukemia and other cancers

  • Standardized IHC protocols using FADD (Ab-191) could help stratify patients for clinical studies

  • Correlation studies between FADD levels and treatment responses may identify predictive markers

• Therapeutic targeting approaches:

  • Monitor FADD expression following experimental therapies

  • Use to validate gene therapy approaches aimed at restoring FADD function

  • Study TAT-FADD conjugates as potential therapeutic proteins for cancer treatment

• Innovative research directions:

  • Investigate FADD in cancer stem cell populations

  • Explore FADD regulation of metabolic pathways in cancer cells

  • Examine FADD interactions with immune checkpoint regulators

What methodological considerations should be addressed when using FADD (Ab-191) Antibody in high-throughput screening or omics research?

For high-throughput applications with FADD (Ab-191) Antibody:

• Assay adaptation considerations:

  • Miniaturization requires antibody revalidation at smaller volumes

  • Determine minimum detectable concentration in high-throughput formats

  • Optimize signal-to-noise ratios for automated detection systems

• Omics integration approaches:

  • Use FADD (Ab-191) for validation of transcriptomic findings

  • Design targeted proteomics assays using epitope-specific peptides

  • Develop multiplexed assays combining FADD with other apoptotic markers

• Quality control requirements:

  • Include position controls on each plate to monitor plate-to-plate variation

  • Establish robust normalization methods for cross-plate comparisons

  • Validate findings with orthogonal methods for selected candidates

What are the recommended dilutions and applications for FADD (Ab-191) Antibody across different experimental procedures?

Data compiled from multiple sources .

What cell lines and tissue samples serve as reliable positive controls for FADD (Ab-191) Antibody validation?

Recommended positive control sources:

• Cell lines:

  • Human: Jurkat, HCT116, HeLa, MCF-7, A549

  • Mouse: RAW 264.7, NIH/3T3

• Tissue samples:

  • Human: Thymus, lymph nodes, spleen

  • Mouse: Thymus, spleen, liver

• Recombinant proteins:

  • Full-length human FADD protein

  • GST-tagged FADD C-terminal domain