Western blot analysis of extracts from Ramos cells (human), untreated or IgM-treated (12 µg/ml for 2 minutes) and mouse spleenocytes, untreated or IgG-treated (30 µg/ml), using Phospho-SHIP1 (Tyr1020) Antibody.
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.
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-SHIP1 (Tyr1021) Antibody detects endogenous levels of SHIP1 only when phosphorylated at tyrosine 1020 of mouse SHIP1 (or Tyr1021 of human SHIP1).Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Tyr1020 of mouse SHIP. Antibodies are purified by protein A and peptide affinity chromatography.
SH2-containing inositol phosphatase 1 (SHIP1) is a hematopoietic phosphatase that hydrolyzes phosphatidylinositol-3,4,5-triphosphate to phosphatidylinositol-3,4-bisphosphate (1). SHIP1 is a cytosolic phosphatase with an SH2 domain in its amino terminus and two NPXY Shc binding motifs in its carboxy terminus (1,2). Upon receptor cross-linking, SHIP is first recruited to the membrane junction through binding of its SH2 domain to the phospho-tyrosine in the ITIM motif (2), followed by tyrosine phosphorylation on the NPXY motif (2). The membrane relocalization and phosphorylation on the NPXY motif is essential for the regulatory function of SHIP1 (3-5). Its effect on calcium flux, cell survival, growth, cell cycle arrest, and apoptosis is mediated through the PI3K and Akt pathways (3-5). Tyr1021 is located in one of the NPXY motifs in SHIP1, and its phosphorylation is important for SHIP1 function (6).
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