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Micropurification of Two Human Cerebrospinal Fluid Proteins by High Performance Electrophoresis Chromatography
Author(s) -
Leone Maria Grazia,
Saso Luciano,
Vecchio Alessandra,
Mo Mengyun,
Silvestrini Bruno,
Cheng C. Yan
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb02156.x
Subject(s) - chromatography , gel electrophoresis , polyclonal antibodies , gel electrophoresis of proteins , chemistry , sodium dodecyl sulfate , amino acid , albumin , peptide sequence , biochemistry , polyacrylamide gel electrophoresis , microbiology and biotechnology , biology , antibody , gene , immunology , enzyme
Using C8 reversed‐phase HPLC in conjunction with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N‐terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A‐l, β 2 ‐microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (M r 28,000) and cerebrin 30 (M r 30,000) that have an N‐terminal amino acid sequence of NH 2 ‐APPAQVSVQPNF and NH 2 ‐APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS‐PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis Chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of >90%. The potential use of this technology for micropurification of proteins was discussed.