Outline of the Biochemical Techniques Used in the Study of ALKPositive ALCL

Biochemical analyses using antibodies have provided valuable additional information on NPM-ALK and variant ALK proteins, their functional activity in vivo, and the identity of interacting proteins, such as PLC-g and PI-3 kinase (23,24). The first step is the solubilization of the proteins from cells under study. Once the proteins have been extracted, they are available for analysis with antibodies using either Western blotting or immunoprecipitation techniques. In Western blotting studies, the proteins are separated on the basis of size by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE). They are then transferred to a membrane, where they can be stained using antibodies in an indirect labeling technique, for example, antibodies to ALK will permit the molecular weight of any ALK proteins to be found and provide evidence of the identity of the ALK fusion protein (Table 1) (25). Immunoprecipitation techniques permit the purification and concentration of specific proteins of interest through the use of antibodies directed against specific proteins being linked onto solid immunoadsorbent beads, for example, Protein G:Gammabind Plus Sepharose. Relevant proteins in solution bind to the specific antibody on the beads and can then be removed from solution by brief centrifugation. The resulting antigen:antibody complexes present in the immunoadsorbent pellet are then separated by SDS-PAGE and identified by Western blotting (26). In an in vitro kinase assay, radiolabeled immunopre-cipitated proteins are detected and identified by autoradiography after SDS-PAGE (25).

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