Western blot analysis of extracts from NK-92 cells, untreated or treated with Human Interleukin-2 (hIL-2) #8907 (10 ng/ml, 15 minutes), using Phospho-Jak3 (Tyr980/981) (D44E3) Rabbit mAb (upper) or Jak3 Antibody #3775 (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-Jak3 (Tyr980/981) (D44E3) Rabbit mAb detects endogenous levels of Jak3 when phosphorylated at Tyr980/981. Some reactivity may exist with single phosphorylation at these sites.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to a region surrounding Tyr980/981 of human and mouse Jak3 protein.
Members of the Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) are activated by ligands binding to a number of associated cytokine receptors (1). Upon cytokine receptor activation, Jak proteins become autophosphorylated and phosphorylate their associated receptors to provide multiple binding sites for signaling proteins. These associated signaling proteins, such as Stats (2), Shc (3), insulin receptor substrates (4), and focal adhesion kinase (FAK) (5), typically contain SH2 or other phospho-tyrosine-binding domains.
Jak3 is primarily expressed in hematopoietic cells and is required for immune cell function and development (6-8). It binds to the common γ subunit (γc) and a shared receptor subunit also used by several cytokines including IL-2, IL-4, IL-7, IL-9, and IL-15 (9). IL-2 signaling and Stat5 activation is highly impaired by the loss of Jak3 (10,11). Jak3 is phosphorylated at multiple sites, including Tyr980 and 981 within its activation loop (12-14).
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