Western blot analysis of extracts from CHO cells transfected with wild-type (WT) or mutant (Y362F) p62Dok, using p62Dok 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 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 June 2020
Protocol Id: 10
p62Dok Antibody detects transfected levels of p62Dok proteins. The antibody does not cross-react with related proteins.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr398 of human p62Dok. Antibodies are purified by protein A and peptide affinity chromatography.
p62Dok (Dok-1) is a major tyrosine-phosphorylated, GAP-associated, 60 kDa protein present within the cells transformed by different tyrosine kinases (1). p62Dok contains an amino-terminal pleckstrin homology domain potentially involved in phospholipid interaction and membrane targeting, a central putative phospho-tyrosine binding domain for interacting with tyrosine-phosphorylated proteins. There are numerous tyrosines in its carboxy-terminal region that are potential targets for tyrosine kinases. If phosphorylated, these tyrosines could serve as docking sites for proteins that contain an SH2 domain (2). Overexpression of p62Dok has been shown to inhibit Ras activity in human embryonic kidney 293 cells and B cell antigen receptor-mediated c-Fos promoter activation in an immature B cell line (3), suggesting that p62Dok may play a negative role in Ras signaling. Moreover, p62Dok overexpression may also inhibit insulin-stimulated Akt activation (4).
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