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Fig. 1. Fractionation of human serum samples. 1DE ( left panel ): 10
g of fractionated
serum eluted either with acetic buffer ( lane b ) or with 2D DIGE lysis buffer ( lane c ) were separated on a 10%-SDS-PAGE
gel. The proteins were stained with a ruthenium-based fl uorescent dye, and protein patterns were visualized by laser
scanning. In contrast to the native serum sample displaying more or less one albumin band after fractionation, a rich band
pattern covering the whole separation range is detectable. No signifi cant difference is detectable between the recom-
mended acetic and 2D DIGE lysis buffer elution. 2DE ( right panel ): 150
μ
g of native human serum ( lane a ) and 10
μ
g of
fractionated serum eluted with 2D DIGE lysis buffer ( right ) were separated in the fi rst dimension by isoelectric focusing
(IEF) (pH 4-7) and in the second dimension on a 12%-SDS-PAGE gel. The proteins were stained with a ruthenium-based
fl uorescent dye, and protein patterns were visualized by laser scanning. Image analysis was performed using
Proteomweaver™ software. Fractionation of the serum samples resulted in a substantial increase of detectable protein
spots. The spot count rose from approximately 1,400 to 2,200 which equates to a 60% increase.
μ
g of native human serum ( left ) and 150
μ
for reproducible sensitive protein profi ling. To minimize sample
loss and experimental error between both methods, the 2D DIGE
lysis buffer was selected for elution of captured proteins. This
enables direct and effective analysis of protein populations that are
usually masked by high-abundance proteins in the native sample
(see Fig. 1 ). In addition, column-to-column reproducibility was
tested to evaluate potential variability being introduced by the
experimental procedure (see Fig. 2 ).
3.1. Serum Samples
1.
Blood samples were exclusively collected in 2.7-mL
S-Monovettes ® (Sarstedt). After sample collection, the
S-Monovettes ® were incubated at room temperature (RT;
25°C) for 30 min.
2.
Incubated samples were centrifuged at 1,400 × g for 10 min
and the supernatant was collected using a polyethylene transfer
pipette.
3.
Finally, samples were aliquoted (1.2 mL) and immediately
transferred to −80°C for long-term storage.
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