Western blot analysis of extracts from Jurkat cells transfected with p56Dok-2 proteins, untreated or stimulated with anti-CD2 antibody, using Phospho-p56Dok-2 (Tyr351) Antibody (upper) or p56Dok-2 Antibody #3914 (lower).
|MW (kDa)||56 to 58|
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-p56Dok-2 (Tyr351) Antibody detects transfected levels of p56Dok-2 only when phosphorylated at tyrosine 351. The antibody does not cross-react with other tyrosine phosphorylated p62Dok family members.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr351 of mouse p56Dok-2. Antibodies are purified by protein A and peptide affinity chromatography.
Docking proteins are substrates of tyrosine kinases that function in the recruitment and assembly of specific signal transduction molecules. There are five members in the p62dok family, p62Dok (Dok-1), p56Dok-2 (Dok-2, or DoK-R), Dok-3, Dok-4 and Dok-5 (1-3), characterized by the presence of an amino-terminal PH domain, a central PTB domain and numerous potential sites of tyrosine phosphorylation. Tyrosine phosphorylation of p56Dok-2 occurs upon stimulation of cells with a variety of stimuli, or in cells transformed by oncogenic tyrosine kinases such as v-Src and Bcr-Abl (3-5). Based on the presence of several signaling domains (PH, PTB domain, tyrosine residue and proline-rich regions), it has been proposed that the p62dok family act as docking proteins that link RTKs to signal transduction pathways. p56Dok-2 has been proposed to be a negative regulator of cytokine-induced proliferation in T cells (5). Phosphorylated Tyr351 of p56Dok-2 mediates an association with the SH2 domain of Nck (4).
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