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Fig. 5. Verification of the filamin-FIP interaction. (A) 20 µg/ml each of filamin rod polypeptides rod 3, rod 4, rod 5-6, rod 2-4, rod 4-6 and an unrelated polypeptide (gst-rez 1) were immobilized on plastic and increasing amounts of FIP polypeptide (rec c-FIP; residues 1612-1907) were added. The ability of the rod proteins to bind the recombinant FIP polypeptide was measured by ELISA using mAb K12-454-2. Measurements were performed in duplicate. Data from a representative experiment are shown. (B) Direct interaction of FIP polypeptide and rod 2-4. Both polypeptides (30 µg each) were mixed and immunoprecipitation was performed using mAb K12-454-2. The immunoprecipitate was resolved by SDS-PAGE (12% acrylamide) and stained with Coomassie Blue (a). The control in c shows that rod 2-4 polypeptide (see b) is not precipitated by mAb K12-454-2 in the absence of the FIP polypeptide. The proteins were separated by SDS-PAGE (10% acrylamide). The location of the molecular weight markers as well as of the immunoglobulin light (lc) and heavy chain (hc) is indicated by arrows. The band at approximately 70 kDa also represents the antibody.
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