Apoptosis occurs not only during programmed cell death, but also during the death process induced by some cytotoxic T cells. A protein ligand, FASL, was identified that triggers cell death by binding to the cell surface receptor variously known as FAS or APT1 family of receptors that includes the 2 tumor necrosis factor (TNF) receptors. The FAS antigen is expressed not only in the cells of the immune system but also in the liver, lung, ovary, and heart, where its function is unclear. FAS ligand/FASL (human 281-aa, ~ 32kD, chromosome 1q23) is a type II transmembrane protein that belongs to the tumor necrosis factor family. FASL is expressed in activated splenocytes and thymocytes, consistent with its involvement in T-cell-mediated. FASL is proteolytically cleaved at the cell surface and relased into the extracellualr fluid. FASL is alternative spliced into two forms: Defects in TNFSF6 are a cause of autoimmune lymphoproliferative syndrome (ALPS), also known as Canale-Smith syndrome (CSS), a childhood syndrome involving hemolytic anemia and thrombocytopenia with massive lymphadenopathy and splenomegaly.
Human FASL-1 (100-178) was expressed in E. colli as 26kD his-tagged fusion protein and purified >95%. For western blot +ve control , it is supplied in SDS-PAGE sample buffer (reduced). Load ~10 ul/lane to visualize with appropriate antibodies. Store at -20°C in suitable aliquots. Avoid repeated thawing or heating.
Applications
Suitable for use in ELISA, Western Blotting. Other applications not tested.
Recommended Dilution
Western Blot: 1:1000-1:5000) for neat serum using ECL. We recommend the use of 0.5-1% milkin diluents used for primary antibody and secondary antibody-enzyme conjugates to suppress background. Rat, mouse and human FASL have 278, 279, and 281aa residues, respectively (Calculated MW of ~32kD). Western blot of the oocyte has detected a specific 31-kD band (1). A 40kD FASL has been detected in mouse T cells (1). An alternate detecting reagent, mFAS-Fc (1) also detected a cell surface protein of about 40kD. ELISA: 1:10,000-50,000 for neat serum and 0.5-1ug/ml for affinity pure. Control peptide can be used to coat ELISA plates at 1ug/ml.
Optimal dilutions to be determined by the researcher.
Control peptide, because of its low MW (<3kD), is not suitable for Western. It should be used for ELISA or antibody blocking experiments (use 5-10 ug control peptide per 1ug of affinity purified IgG or 1ul antiserum) to confirm antibody specificity.
Storage and Stability
May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Immunogen
13-aa peptide from human FASL
Form
Supplied as a liquid, 0.05% sodium azide.
Specificity
94% conserved in mouse. It is poorly conserved in human FASL. We recommend using antibody for human FASL.
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