Western blot analysis of extracts from serum-starved BaF3 cells or serum-starved BaF3 cells stably expressing mouse thrombopoietin receptor (mTPOR), untreated (-) or treated with recombinant mouse thrombopoietin (mTPO; 100 ng/mL, 10 min; +), using Phospho-TPOR (Tyr626) (D3H7B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from serum-starved M-07e cells, untreated (-) or treated with recombinant human thrombopoietin (hTPO; 100 ng/mL, 10 min; +), using Phospho-TPOR (Tyr626) (D3H7B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (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-TPOR (Y626) (D3H7B) Rabbit mAb recognizes endogenous levels of TPOR protein only when phosphorylated at Tyr626. This antibody cross-reacts with a 30 kDa protein of unknown origin. This antibody may cross-react with some tyrosine-phosphorylated proteins including the EGF Receptor, ROS1, and FLT3.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey, Bovine
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr626 of human TPOR protein.
Thrombopoietin receptor (TPOR, c-Mpl) is a hematopoietic receptor that binds the growth factor, thrombopoietin (TPO), responsible for regulation of platelet production (1-3). Expression of TPOR by megakaryocytes is required for megakaryocyte growth and development (4). TPOR is also expressed by hematopoietic stem cells and is required for stem cell maintenance and expansion (5). Studies show that mice lacking either TPOR or TPO have severely reduced numbers of platelets and megakaryocytes as well as decreased numbers of other hematopoietic lineages (4,5). Binding of TPO to TPOR induces receptor dimerization that leads to phosphorylation and activation of the tyrosine kinase Jak2. Activated Jak2 associates with the cytoplasmic domain of TPOR (6,7) and phosphorylates TPOR at Tyr626 and Tyr631 (8). These phosphorylated tyrosine residues provide docking sites for downstream signaling molecules including Stat3, Stat5, Shc, and SHIP (7-9).
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