Environmental Engineering Reference
In-Depth Information
haptoglobin were found to be more resistant to the higher temperatures tested, as
they undergo changes in abundance only at room temperature; conversely, other
spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as
they undergo changes in abundance at all temperatures except at
80
C.
15.2.4 Sample Preparation Method
Samples should have a high protein concentration (
>
10 mg/ml) and be free of salt
and other disturbing factors, such as ionic detergents, nucleic acids, lipids, etc. that
may interfere with the 2-DE. Precipitation followed by pellet uptake in isoelectric
focusing (IEF) compatible sample solution is employed to concentrate and selec-
tively separate proteins in the sample from the contaminating species. Protein
concentration and desalting methods, however, are associated with artefacts,
which can affect the results of the proteomic study, the most common being the
incomplete protein recovery. Thus, the protein sample preparation method plays an
important role in deducing the maximum information from 2-DE studies. Various
methods like the ultrafiltration, precipitation using acetone, TCA, acetone/TCA,
chloroform/methanol, ammonium sulphate have been studied to note their influence
on protein resolution in 2-DE analysis [
58
]. In general, the precipitation methods
and ultrafiltration delivered comparable results with no profound differences
observed between untreated plasma and treated plasma samples. A relatively easy
to perform concentration and desalting step using acetone precipitation resulted in a
good recovery of all protein spots. Quantitative ammonium sulphate precipitation
also resulted in an efficient precipitation of most proteins though spots representing
certain proteins, like complement factor B, C3a and clusterin, were weak. The
chloroform/methanol method yielded satisfactory results as well, although the spots
representing C3a and clusterin are missing in the precipitate. Ultrafiltration resulted
in recovery of practically all proteins. Thus, TCA precipitation, acetone precipita-
tion and ultrafiltration delivered a higher protein recovery compared to ammonium
sulphate and chloroform/methanol steps. Based on ease of performance, time
required and cost effectiveness, precipitation using TCA/acetone was found to be
the best method [
59
].
15.2.5 Surface Charge
Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of
biopolymers, like proteins, organised into the “protein corona” that is associated
with the nanoparticle and continuously exchanging with the proteins in the envi-
ronment. Lundquist et al., have used six different polystyrene nanoparticles to study
three different surface chemistries (plain polystyrene—neutral, carboxyl-modi-
fied—negatively charged, and amine-modified—positively charged) and two
