Western blot analysis of extracts from Jurkat, 32D and BaF3 cells using SHP-1 (C14H6) Rabbit mAb.
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
SHP-1 (C14H6) Rabbit mAb detects endogenous level of total SHP-1 protein.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro161 of human SHP-1.
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).
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.