Western blot analysis of extracts from COS cells, untransfected or overexpressing Myc-tagged Ack1 protein, using Phospho-Ack1 (Tyr284) Antibody (upper and middle) and Myc-Tag (71D10) Rabbit mAb #2278 (lower). The middle blot was treated with calf intestinal phosphatase (CIP) before antibody probing. (The lysates of COS cells overexpressing Myc-tagged Ack1 were kindly provided by Dr. Wannian Yang, Weis Center for Research, Geisinger Clinic.)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 and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-Ack1 (Tyr284) Antibody detects transfected levels of Ack1 only when phosphorylated at Tyr284.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr284 of human Ack1. Antibodies are purified by peptide affinity chromatography.
Ack1 and Ack2 (activated cdc42-associated kinase 1 and 2) are non-receptor tyrosine kinases that consist of a tyrosine kinase core, an SH3 domain, a cdc42/Rac-binding (CRIB) domain, a Ralt homology region and a proline-rich region (1,2). Ack1 and 2 are the only two tyrosine kinases known to interact with cdc42. Both Acks are activated by growth factors including EGF and PDGF, as well as by activated integrins through cell adhesion, and may serve to link receptor tyrosine kinase or G protein-coupled receptor signaling with cdc42. Acks may regulate cell growth, morphology and motility (3,4). Recent findings indicate that Ack1 may play a role in prostate tumorigenesis, making it a potential drug target for this type of cancer (5).
Tyr284 is located in the activation loop of Ack1 and is a primary autophosphorylation site critical for Ack1 kinase activity (5).
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