Western blot analysis of extracts from serum-starved U266 and Jurkat cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (50 ng/ml, 15 min; +) using Phospho-Tyk2 (Tyr1054/1055) (D7T8A) Rabbit mAb (upper) or total Tyk2 (D4I5T) Rabbit mAb #14193 (lower).
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 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-Tyk2 (Tyr1054/1055) (D7T8A) Rabbit mAb recognizes endogenous levels of Tyk2 protein only when phosphorylated at Tyr1054 and Tyr1055. Cross-reactivity was not observed with other Jak family members.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Tyr1054/1055 of human Tyk2 protein.
Tyk2 is a member of the Jak family of protein tyrosine kinases. It associates with and is activated by receptors for many cytokines including IL-13, the IL-6 family, IL-10, and IFN-α and β (1-3). Following ligand binding, Tyk2 is activated by phosphorylation of Tyr1054 and/or Tyr1055 (4). Tyk2 is required for the tyrosine phosphorylation of Stat3 in the IFN-β signaling cascade (5).
The role of Tyk2 has been extensively studied in terms of its involvement in immune regulation and pathological significance (reviewed in 6). Deletion of Tyk2 in mice results in increased sensitivity to infection and defective tumor surveillance, but only a partial effect on Type I interferon signaling (7, 8). In contrast, a human patient diagnosed with hyper-IgE syndrome having increased susceptibility to various microorganisms was found to have a homozygous mutation of Tyk2 (9). These studies suggest a more critical role of Tyk2 in humans with regards to Type I interferon signaling as well as other cytokines including IL-23, IL-6, and IL-10.
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