Western blot analysis of extracts from Sup-M2 and Karpas-299 cells using Phospho-ALK (Tyr1278) Antibody (upper and middle) and ALK (C26G7) Rabbit mAb #3333 (lower). The middle and lower blots were treated with calf intestinal phosphatase to dephosphorylate NPM-ALK. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
|MW (kDa)||220 kDa (ALK), 80 kDa (NPM-ALK)|
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
Phospho-ALK (Tyr1278) Antibody detects ALK only when phosphorylated at Tyr1278, which is equivalent to Tyr338 of NPM-ALK. This antibody also reacts with leukocyte tyrosine kinase (LTK) phosphorylated at Tyr672, and may cross-react with other activated protein tyrosine kinases including Bcr-Abl.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr1278 of human ALK. Antibodies are purified by protein A and peptide affinity chromatography.
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor for pleiotrophin (PTN), a growth factor involved in embryonic brain development (1-3). In ALK-expressing cells, PTN induces phosphorylation of both ALK and the downstream effectors IRS-1, Shc, PLCγ, and PI3 kinase (1). ALK was originally discovered as a nucleophosmin (NPM)-ALK fusion protein produced by a translocation (4). Investigators have found that the NPM-ALK fusion protein is a constitutively active, oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Research literature suggests that activation of PLCγ by NPM-ALK may be a crucial step for its mitogenic activity and involved in the pathogenesis of anaplastic lymphomas (5).
A distinct ALK oncogenic fusion protein involving ALK and echinoderm microtubule-associated protein like 4 (EML4) has been described in the research literature from a non-small cell lung cancer (NSCLC) cell line, with corresponding fusion transcripts present in some cases of lung adenocarcinoma. The short, amino-terminal region of the microtubule-associated protein EML4 is fused to the kinase domain of ALK (6-8).
Phosphorylation of ALK on Tyr1278 was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for phosphorylation site discovery. Phosphorylation of ALK at Tyr1278 was observed in select carcinoma cell lines and in tumors (6).
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