Biomedical Engineering Reference
In-Depth Information
second dimension according to their size (molecular weight) by SDS-PAGE. This
technique has been optimized for the analysis of adsorbed plasma proteins on
polystyrene particles, solid lipid nanoparticles (SLNP), and magnetite NP [
18
].
After the proteins are separated, a staining method must be used so that the
protein spots can be visualized in the gel and then converted into a digital image. An
ideal staining method should be sensitive enough to detect low-abundance proteins
and have a linear dynamic range throughout all the spots of different intensity in the
gel. Moreover, the spot of interest can be excised from the gel and analyzed in order
to obtain the identities of the protein spots.
Blue Coomassie staining suffers from low sensitivity. Ammoniac silver staining
and several of the fluorescent approaches ensure high sensitivity and detecting less
that 1 ng of proteins. Another attractive approach is given by difference in gel
electrophoresis (DIGE) that requires the use of cyanine fluorophore dyes (Cy dyes)
that covalently bind proteins in a complex mixture prior to the IEF and 2D-PAGE.
A DIGE experiment requires the label of each individual sample with one Cy dye.
After the protein migration is performed, the gel is scanned using a fluorescent
scanner obtaining different images from the same gel using emission and excitation
settings for each of the dye. This approach is particularly attractive because, if in the
same gel two sample conditions are run, it is possible to identify spot changes
within the same gel, thereby reducing any error that is caused by running samples in
different gels. After image capture, the gel images have to be imported into image
analysis software which quantifies the spot intensity and compares spot abundance
from different gels, and the matching of the same spots across the gels can be quite
challenging. Gel spots that are significantly different between gels can be analyzed
afterwards by mass spectrometry to obtain protein identity.
Gessner et al. [
19
] investigated changes in the plasma protein adsorption patterns
as a function of surface hydrophobicity. Latex particles with decreasing surface
hydrophobicity were synthesized as model colloidal carriers. Physicochemical
characterization has been performed and considerable differences in the protein
adsorption patterns on the particles could be detected by using 2D-PAGE.
Although 2D technique provides a reproducible protein migration in a gel and
protein identities can be obtained relatively quick, obtaining high gel quality can be
quite time consuming and have several technical difficulties; it requires specific
equipments. Especially if using fluorescent-stained approaches, the image analysis
to obtain spot changes within gels is quite expensive.
G¨ppert et al. [
20
] studied the NP-plasma protein interaction using 2D-GE
approaches and showed that the protein adsorption pattern on solid lipid NP was
dependent on incubation time. This study confirmed the Vroman effect that
explains the time evolution of the corona. According to the Vroman effect, proteins
with high affinities but in low concentration in the plasma can displace highly
concentrated proteins that have low affinity for the NPs.
2D-PAGE is a common and powerful method for separation of the proteins of
the corona. The data usually is compared with a master map of human plasma
protein. However, the challenge is how to find a master protein map. The human
plasma protein map can be different by changing the plasma donor or the
