Western blot analysis of extracts from various cell lines, untreated (-) or treated (+) with λ phosphatase, using Phospho-SHP-1 (Tyr564) (D11G5) Rabbit mAb (upper) and SHP-1 (C14H6) Rabbit mAb #3759 (lower). The JCaM1.6 cell line is a Lck kinase-deficient derivative of the Jurkat cell line. Note the corresponding reduction in phospho-SHP-1 (Tyr564) protein in JCaM1.6 cells, relative to wild-type (Jurkat) cells.
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-SHP-1 (Tyr564) (D11G5) Rabbit mAb recognizes endogenous levels of SHP-1 protein only when phosphorylated at Tyr564.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr564 of human SHP-1 protein.
SHP-1 (PTPN6) is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells. The enzyme is composed of two SH2 domains, a tyrosine phosphatase catalytic domain, and a carboxy-terminal regulatory domain (1). SHP-1 removes phosphates from target proteins to downregulate several tyrosine kinase-regulated pathways. In hematopoietic cells, the amino-terminal SH2 domain of SHP-1 binds to tyrosine phosphorylated erythropoietin receptors (EpoR) to negatively regulate hematopoietic growth (2). Overexpression of SHP-1 in epithelial cells results in dephosphorylation of the Ros receptor tyrosine kinase and subsequent downregulation of Ros-dependent cell proliferation and transformation (3). Following ligand binding in myeloid cells, SHP-1 associates with the IL-3R β chain and downregulates IL-3-induced tyrosine phosphorylation and cell proliferation (4). Because SHP-1 downregulates various proliferation pathways, SHP-1 is considered a potential tumor suppressor and angiogenesis regulator (5,6).
SHP-1 is a substrate of Src family kinases (7,8) and phosphorylation of Tyr564 is thought to be critical for achieving maximal phosphatase activity (8). In a murine model of chronic myelomonocytic leukemia (CMML), genetic suppression of Tyr564 phosphorylation led to constitutive overactivation of the transcription factor Stat5 and an accelerated onset of CMML-like disease (8).
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