Determination of plasma protein adsorption on magnetic iron oxides: sample preparation.
OBJECTIVE
The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).


METHODS
The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2).


RESULTS
All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5-7.5.


CONCLUSIONS
Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.
Thode
K
K
Institut für Pharmazeutische Technologie, Biopharmazie und Biotechnologie, Freie Universität Berlin, Germany.
Lück
M
M
Semmler
W
W
Müller
R H
RH
Kresse
M
M
D000327:Adsorption; D001798:Blood Proteins / Q000378:metabolism*; D002498:Centrifugation; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D005290:Ferric Compounds / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D008280:Magnetics; D015203:Reproducibility of Results
D001798:Blood Proteins; D005290:Ferric Compounds; C000499:ferric oxide