Western blot analysis of extracts from BaF3 cells, untreated or treated with IL-3 (5 minutes), using Phospho-Jak2 (Tyr221) Antibody #3774 (top) or total Jak2 (D2E12) XP® Rabbit mAb #3230 (bottom).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-Jak2 (Tyr221) Antibody detects endogenous levels of Jak2 only when phosphorylated at Tyr221.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr221 of human Jak2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
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.
Jak2 is autophosphorylated at Tyr1007/1008 in the putative activation loop during cytokine signaling (6). Tyr221 and 570 have also been shown to be prominent sites for autophosphorylation (7,8). Mutational analysis suggests that phosphorylation at Tyr221 may increase kinase activity, while phosphorylation at Tyr570, which lies within the JH2 inhibitory domain, may contribute to inhibiting Jak2 activity. In addition, Tyr813 was identified as a site for autophosphorylation critical for the activation of Jak2 by the SH2 domain-containing protein SH2-B β (9).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Jak antibodies produced under license (granting certain rights including those under U.S. Patent No. 5,658,791) from Chemicon International, Inc.
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