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
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around serine 967 of human ASK1. Antibodies are purified by protein A and peptide affinity chromatography.
Apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, plays essential roles in stress-induced apoptosis (1,2). ASK1 is activated in response to a variety of stress-related stimuli through distinct mechanisms and activates MKK4 and MKK3, which in turn activate JNK and p38 (3). Overexpression of ASK1 activates JNK and p38 and induces apoptosis in several cell types through signals involving the mitochondrial cell death pathway. Embryonic fibroblasts or primary neurons derived from ASK1-/- mice are resistant to stress-induced JNK and p38 activation as well as cell death (4,5). Phosphorylation at Ser967 is essential for ASK1 association with 14-3-3 proteins and suppression of cell death (6). Oxidative stress induces dephosphorylation of Ser967 and phosphorylation of Thr845 in the activation loop of ASK1, both of which are correlated with ASK1 activity and ASK1-dependent apoptosis (7,8). Akt phosphorylates ASK1 at Ser83, which attenuates ASK1 activity and promotes cell survival (9).
Phosphorylation at Ser967 of ASK1 leads to association of 14-3-3 and suppression of ASK1 kinase activity (6).
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