REACTIVITY | SENSITIVITY | MW (kDa) | SOURCE |
---|---|---|---|
H | Transfected Only | 155 | Rabbit |
Western blot analysis of extracts from COS cells, untransfected (lane 1), or transfected with wild-type ASK1 (lanes 2,3), using Phospho-ASK1 (Thr845) Antibody (upper) or ASK1 Antibody #3762 (lower). In lane 3, the lysate was treated with lamda phosphatase to demonstrate phospho-specificity of Phospho-ASK1 (Thr845) Antibody.
Learn more about how we get our images.Western blot analysis of extracts from HEK293 cells, untransfected (lane 1) or transfected with wild-type ASK1 (lane 2) or T845A mutant ASK1 (lane 3), using Phospho-ASK1 (Thr845) Antibody (upper) or ASK1 Antibody #3762 (lower). (Lysates were provided by Dr. Wang Min, Dept. of Pathology, Yale University, New Haven, CT).
Learn more about how we get our images.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 datasheet or 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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-ASK1 (Thr845) Antibody detects transfected ASK1 only when phosphorylated at threonine 845.
Human
Mouse
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr845 of mouse 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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Explore pathways related to this product.
Product # | Size | Price |
---|---|---|
3765S | 100 µl | N/A |
Hamilton House
Mabledon Place
London, WC1H 9BB
UK
Need information for a different country? Please click here.
To get local purchase information on this product, click here.