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Fig. 1. Characterization of anti-ALK monoclonal antibody. (A) Preparation of immunogen (APmH). The entire extracellular domain (ECD) of mouse ALK (ALK) followed by a PreScission protease site and a myc-His tag (PmH) was expressed and collected as described in Materials and Methods. The ECD of ALK fused with the Fc portion of human immunoglobulin (ALK-Fc) was used as an antigen for ELISA screening. Abbreviations: SS, signal sequence; TM, transmembrane domain; PTK, protein tyrosine kinase domain. (B) HEK 293T cells were transfected with APmH-expressing vector (+) and the recombinant protein was collected on the chelating Sepharose, separated by SDS-PAGE, and transferred to PVDF membranes for immunoblotting. Supernatant from cells transfected with empty vector was treated in the same way as a negative control (–). The membranes were blotted with either anti-myc (myc) or anti-ALK monoclonal (mAb16-39) antibodies (2 µg/ml). (C) APmH protein was immunoprecipitated with anti-myc antibody (myc), mAb16-39 (16-39), or normal rat IgG (IgG) (2 µg each). As a control, the APmH protein was collected by the chelating Sepharose (HIS), which allowing the purification of the recombinant protein through binding to the His tag epitope. Immunoprecipitated proteins were probed with anti-myc antibody 9E10 (myc).
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