Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.
A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.
Kellogg
D R
DR
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Alberts
B M
BM
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002502:Centrioles / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000737:chemistry* / Q000196:embryology; D004591:Electrophoresis, Polyacrylamide Gel; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008870:Microtubules / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013194:Staining and Labeling
D000906:Antibodies; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA