Separation of human cerebrospinal fluid proteins by capillary isoelectric focusing in the absence of denaturing agents.
Conditions of capillary isoelectric focusing (CIEF) to separate human cerebrospinal fluid (CSF) proteins were examined referring to those which we have established for the separation of human plasma/serum proteins. Since the average protein concentration in CSF is about 1/200 of plasma and the salt concentration is at almost the same level as plasma, desalting of CSF samples with minimum dilution was a prerequisite for CIEF analysis of CSF proteins. We constructed an apparatus to dialyze CSF at the level of 20-30 microL, this volume being sufficient for 3-4 repeated analyses of the CSF sample. To trace the process of dialysis, a simple device to measure the conductivity of the dialyzate was also constructed. Most of the CIEF conditions for plasma protein analysis could be applied for CSF protein analysis. However, the addition of N,N,N',N'-tetramethylethylenediamine (TEMED) at a suitable concentration was necessary to improve the resolution of basic proteins (IgG region), since some CSF patterns showed peaks of basic proteins which are not obvious in the serum of the same patient. About 70 peaks and shoulders of CSF proteins could be detected by the established CIEF technique. The results of CIEF analysis of CSF samples suggested that the technique will be useful as a survey method to detect specific proteins in CSF, which might relate to disorders in the central nervous system.
Manabe
T
T
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan. manabet@dpc.ehime-u.ac.jp
Miyamoto
H
H
Inoue
K
K
Nakatsu
M
M
Arai
M
M
D001798:Blood Proteins / Q000302:isolation & purification; D002556:Cerebrospinal Fluid Proteins / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D011489:Protein Denaturation
D001798:Blood Proteins; D002556:Cerebrospinal Fluid Proteins