Isolation of thrombospondin released from thrombin-stimulated human platelets by fast protein liquid chromatography on an anion-exchange Mono-Q column.
Thrombospondin, a glycoprotein found in human platelet alpha granules, is thought to play a major role in platelet haemostatic functions. A rapid method to isolate thrombospondin for functional and structural studies was developed. Freshly prepared supernatants from thrombin-stimulated platelets were separated on an anion-exchange Mono-Q column on a fast protein liquid chromatography system. Detection of thrombospondin in the eluted peaks was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with silver staining and a solid-phase radioimmunoassay with monoclonal antibodies directed against thrombospondin and other platelet granule glycoproteins. Thrombospondin was isolated rapidly to a high degree of purity using the fast protein liquid chromatography Mono-Q system (20 min), compared with the time taken with other techniques.
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D005353:Fibronectins / Q000032:analysis; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods; D012685:Sepharose / Q000031:analogs & derivatives; D012967:Sodium Dodecyl Sulfate; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology*; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D006023:Glycoproteins; D019699:Thrombospondins; C037405:heparin-sepharose; D012967:Sodium Dodecyl Sulfate; D012685:Sepharose; D013917:Thrombin