|H M||Endogenous||125||Rabbit IgG|
Western blot analysis of extracts from BaF3 cells, untreated or treated with Mouse Interleukin-3 (mIL-3) #8923 (10 ng/ml, 5 min), using Phospho-Jak2 (Tyr1008) (D4A8) Rabbit mAb (upper) or total Jak2 (D2E12) XP® Rabbit mAb #3230 (lower).Learn more about how we get our images.
Western blot analysis of extracts from SET-2 cells, untreated or treated with the Jak2 inhibitor FLLL31 (10 μM, 4 hr), using Phospho-Jak2 (Tyr1008) (D4A8) Rabbit mAb (upper) or total Jak2 (D2E12) XP® Rabbit mAb #3230 (lower).Learn more about how we get our images.
Western blot analysis of extracts from LNCaP cells, untreated or treated with growth hormone (GH) (100 ng/ml, 5 min), using Phospho-Jak2 (Tyr1008) (D4A8) Rabbit mAb (upper) or total Jak2 (D2E12) XP® Rabbit mAb #3230 (lower).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, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Phospho-Jak2 (Tyr1008) (D4A8) Rabbit mAb recognizes endogenous levels of Jak2 protein only when phosphorylated at Tyr1008. This antibody also reacts with Jak2 when dually phosphorylated at Tyr1007 and Tyr1008. Cross-reactivity was not observed with other Jak family members by western blot.
Rat, Monkey, Xenopus, Bovine, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr1008 of human Jak2 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.
Jak2 signaling is associated with a number of cytokines, growth factors, and hormones including IL-3, IL-5, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO) growth hormone, prolactin, and leptin (6-13). The oncogenic potential of Jak2 has been realized though translocations and point mutations resulting in its enhanced, de-regualated kinase activity, making Jak2 a potential therapeutic target. Jak2 gene translocations resulting in fusions with the TEL (TEL-JAK2) and PCM1 (PCM1-JAK2) have been found in leukemia patients (14,15). An activating point mutation in Jak2 resulting in a valine to phenylalanine switch at position 617 (V617F) has been implicated in myeloproliferative disorders including polycythemia vera and essential thrombocythemia (16).
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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