Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.
Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.
Schlaf
G
G
Department of Neurology, University of Göttingen, Germany.
Göddecke
M
M
Wolff
J R
JR
Felgenhauer
K
K
Mäder
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001923:Brain Chemistry; D002138:Calibration; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007525:Isoelectric Focusing; D012394:Rosaniline Dyes; D013552:Swine; D013572:Synaptic Vesicles / Q000502:physiology; D016708:Synaptophysin / Q000032:analysis* / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D012394:Rosaniline Dyes; D016708:Synaptophysin; C048139:Coomassie blue