Western blot analysis of extracts from COS cells, untransfected or transfected with rat Tpl2 , using Phospho-Tpl2 (Ser400) Antibody (upper) and Tpl2 Antibody #4492 (lower). In lane 3, Tpl2 transfected COS cell lysate was treated with λ phosphatase to demonstrate the phospho-specificity of Phospho-Tpl2 (Ser400) Antibody.
|MW (kDa)||60, 62|
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.
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
Phospho-Tpl2 (Ser400) Antibody detects transfected Tpl2 only when phosphorylated at Ser400. This antibody does not cross-react with other phosphorylated MAP kinase kinase kinases.Species Reactivity:
RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a mixture synthetic phosphorylated peptides corresponding to residues surrounding Ser400 of human, mouse and rat Tpl2. Antibodies are purified by protein A and peptide affinity chromatography.
Tpl2 (tumor progression locus 2), also known as COT (cancer osaka thyroid), is a serine/threonine kinase expressed primarily in hematopoietic tissues, lung and liver (1). Over-expression of Tpl2 potentiates MAP kinase pathways through MEK1 and SEK1, as well as through MKK6 and MEK5 (2,3). Tpl2 is also engaged in NF-κB activation through NF-κB inducing kinase (NIK), or by inducing phosphorylation and degradation of the NF-κB precusor, p105 NF-κB1 (4,5). Ser400 of Tpl2 is phosphorylated in an Akt-dependent manner. This phosphorylation is required for Tpl2-induced NF-κB-dependent transcription (6). Tpl2 also activates caspase-3 by promoting the assembly of a protein complex of Apaf1 (apoptotic protease-activating factor 1), caspase-9, Tpl2, adaptor protein Tvl1 and procaspase-3 (7).
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