Western blot analysis of extracts from 293 IL-1R cells treated with IL-1alpha (20 ng/ml) or IL-1beta (10 ng/ml) and Calyculin A (50 nM) for 10 minutes, using Phospho-TAK1 (Thr184/187) Antibody.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: 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
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-TAK1 (Thr184/187) Antibody detects endogenous levels of TAK1 only when phosphorylated at both threonine 184 and threonine 187. This antibody weakly cross-reacts with TAK1 singly phosphorylated at threonine 184.
Mouse, Rat, Chicken, Xenopus, Zebrafish, Bovine
Polyclonal antibodies are produced by immunizing animals with a phosphopeptide corresponding to residues surrounding Thr184 and Thr187 of human TAK1. Antibodies are purified by protein A and peptide affinity chromatography.
TAK1 is a mitogen-activated protein kinase kinase kinase that can be activated by TGF-β, bone morphogenetic protein and other cytokines including IL-1 (1,2). In vivo activation of TAK1 requires association with TAK1 binding protein 1 (TAB1), which triggers phosphorylation of TAK1 (3,4). Another adaptor protein, TAB2, links TAK1 with TRAF6 and mediates TAK1 activation upon IL-1 stimulation (5). Once activated, TAK1 phosphorylates MAPK kinases MKK4 and MKK3/6, which activate p38 MAPK and JNK, respectively. In addition, TAK1 activates the NF-κB pathway by interacting with TRAF6 and phosphorylating the NF-κB inducing kinase (NIK) (2).
TAK1 activation requires multiple phosphorylations in its activation loop. Mutations of Thr187 and Thr184, residues located in the activation loop of TAK1, impairs phosphorylation of both TAK1 and TAB1 and reduces the kinase activity of TAK1, suggesting that autophosphorylation of these residues is necessary for TAK1 activation (4).
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