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Optimising ovine cerebrospinal fluid preparation for two‐dimensional gel electrophoresis
Author(s) -
Chen Ruo Li,
Sage Elizabeth A.,
Dunn Michael J.,
Wait Robin,
Preston Jane E.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500623
Subject(s) - transthyretin , cerebrospinal fluid , proteome , chromatography , two dimensional gel electrophoresis , chemistry , gel electrophoresis , acetone , tetramer , sample preparation , proteomics , microbiology and biotechnology , biology , biochemistry , pathology , medicine , enzyme , gene
Biomarkers for neurodegenerative disorders are potentially present in cerebrospinal fluid (CSF) and can be detected using proteomic technologies. Since CSF is high in salt and low in protein, its study by proteomic methods requires appropriate sample preparation. In this study, we applied four different sample treatments to the same ovine CSF sample. Precipitation with acetone or using a 2‐D Clean‐Up Kit (GE Healthcare BioSciences, Little Chalfont, UK) preserved more proteins, and produced more gel spots than spin columns from Sigma and Bio‐Rad. A 53‐kDa spot, identified by MS/MS as transthyretin (TTR) tetramer, was not detected in samples treated with the 2‐D Clean‐Up Kit, though it was always present on all gels prepared using the other three methods. Western immunoblotting confirmed the low recovery of tetrameric TTR by the 2‐D Clean‐Up Kit and showed that the tetrameric form of TTR predominated in ovine but not in rat CSF. In one ovine CSF sample haemoglobin was found, indicating blood contamination. We conclude that acetone precipitation is a simple and efficient way to prepare ovine CSF for 2‐DE. The use of the 2‐D Clean‐Up Kit leads to the disappearance of tetrameric TTR only from ovine CSF proteome.