Western blot analysis of NK-92 cells, untreated (-) or treated (+) with TPA #4174 (80 nM, 1 hr) and Ionomycin #9995 (3 μM, 1 hr) followed by Brefeldin A #9972 (300 ng/ml, 4 hr), using FasL (D1N5E) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot anlaysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human FasL (hFasL-Myc; +), using FasL (D1N5E) Rabbit mAb.
Western blot analysis of extracts from recombinant HumanHis6 Fas Ligand/TNFSF6 (hHis6FasL) #5452 using FasL (D1N5E) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
FasL (D1N5E) Rabbit mAb recognizes endogenous levels of total FasL protein.
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human FasL protein. The antigen is within the extracellular domain of FasL.
Association of the receptor Fas with its ligand FasL triggers an apoptotic pathway that plays an important role in immune regulation, development, and progression of cancers (1,2). Loss of function mutation in either Fas (lpr mice) or FasL (gld mice) leads to lymphadenopathy and splenomegaly as a result of decreased apoptosis in CD4-CD8- T lymphocytes (3,4). FasL (CD95L, Apo-1L) is a type II transmembrane protein of 280 amino acids (runs at approximately 40 kDa upon glycosylation) that belongs to the TNF family, which also includes TNF-α, TRAIL, and TWEAK. Binding of FasL to its receptor triggers the formation of a death-inducing signaling complex (DISC) involving the recruitment of the adaptor protein FADD and caspase-8 (5). Activation of caspase-8 from this complex initiates a caspase cascade resulting in the activation of caspase-3 and subsequent cleavage of proteins leading to apoptosis. Unlike Fas, which is constitutively expressed by various cell types, FasL is predominantly expressed on activated T lymphocytes, NK cells, and at immune privileged sites (6). FasL is also expressed in several tumor types as a mechanism to evade immune surveillance (7). Similar to other members of the TNF family, FasL can be cleaved by metalloproteinases producing a 26 kDa trimeric soluble form (8,9).
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