|H||Endogenous||80 (NPM-ALK), 220 (ALK)||Rabbit IgG|
Immunoprecipitation of Karpas-299 cell lysates using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 and ALK (C26G7) Rabbit mAb (Sepharose® Bead Conjugate). The western blot was probed using ALK (31F12) Mouse mAb #3791. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.Learn more about how we get our images.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
ALK (C26G7) Rabbit mAb (Sepharose® Bead Conjugate) detects endogenous levels of total ALK protein. This antibody does not cross-react with other family members.
Monoclonal antibody is produced by immunizing animals with a recombinant fusion protein surrounding amino acid 1475 of human ALK.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. ALK (C26G7) Rabbit mAb (Sepharose® Bead Conjugate) is useful for immunoprecipitation assays. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated ALK (C26G7) Rabbit mAb #3333.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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