Isolation of platelet microtubule protein by an immunosorptive method.
A rapid and highly specific method for the isolation of human platelet tubulin by immunosorption was developed. Platelet tubulin isolated by successive cycles of polymerization was used as antigen. Densitometric quantification of the antigen subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 96% tubulin of molecular weight 55,000 and 4% high molecular weight proteins (mr = 240,000 to 250,000) which co-purified with platelet micrtobule protein. Platelet tubulin bound 0.57 mumol of colchicine/100 mg of protein. Monospecific antibody of human platelet tubulin was prepared in rabbits. After absorption with tubulin co-purifying high molecular weight proteins, and serum proteins, the rabbit anti-tubulin serum gave a single precipitin line on double immunodiffusion against platelet tubulin and the high speed supernatant of a platelet sonicate (platelet extract). The antiserum precipitated the colchicine-binding activity of platelet extracts. The gamma-globulin fraction of the absorbed antiserum was linked to an agarose matrix. Platelet extracts applied to such immunosorptive columns showed the disappearance of a single protein which was eluted with 0.5 g/liter of Triton X-100 and identified as platelet tubulin. Its colchicine-binding activity was retained in full. Electron microscopic examination revealed that the ability of platelet tubulin to polymerize and form tubules was not impaired in the presence of 0.5 g/liter of Triton X-100. This simple isolation procedure of platelet tubulin has great advantages in terms of purity and yield and can readily be adapted for use with other cell systems.
D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000032:analysis* / Q000648:ultrastructure; D008970:Molecular Weight; D011092:Polyethylene Glycols; D011485:Protein Binding; D011487:Protein Conformation; D014404:Tubulin / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification
D006023:Glycoproteins; D046911:Macromolecular Substances; D014404:Tubulin; D011092:Polyethylene Glycols