Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 45)

In preparative 2-D electrophoresis the first step fractionates proteins into
defined pH ranges by liquid-phase isoelectric focusing in the Rotofor cell. The
Rotofor is capable of 500-fold, or more, purifications of proteins from complex
mixtures. Proteins are concentrated in discrete liquid fractions at their respective
isoelectric points. In the second purification step, preparative polyacrylamide gel
electrophoresis (PAGE) in the Model 491 Prep Cell, individual proteins are isolated
on the basis of their size differences.
Samples such as plasma pose a particular problem for electrophoretic
techniques due to the presence of high concentrations of albumin immunoglobulins,
which together make up more than 65% of the total plasma protein. The high
protein load severely limits the volume of plasma that can be purified by
conventional electrophoretic means. The preparative-2D method circumvents this
problem. The method is illustrated with purification of a 70 kd dimeric (34 and 36 kd)
apolipoprotein (Apo J) and a 49 kd uncharacterized protein which in previous
blotting experiments appeared to have a blocked amino-terminus. Both have
glycosylated isoforms with pIs ranging from 4.9 tom 5.3. Apo J which is in the
0.05 mg/ml concentration range represents less than 0.15% of total plasma protein.
The low plasma concentration of Apo J and the 49kd uncharacterized protein is
evident from analytical 2-D PAGE of whole plasma (Figure 1) where they are barely
visible with silver staining.
13.2 Methods
Analytical 2-D PAGE
5 microliters of whole human plasma were diluted with 10 microliters of
dithioerythritol (DTE, 1% w/v), containing SDS (10% w/v). After a 5 minute incubation
at 95 ÞC the sample was diluted to 500 microliters with DTE (1% w/v), CHAPS
(4% v/v), urea (9M) and ampholytes (pH range 3-11, 5% v/v). Aliquots of 30 microliters
(containing 18 micrograms of protein) were used for analysis on 2-D gels. See
Figure 1. Protein containing fractions obtained from the Rotofor cell were similarly
treated.
Sample preparation for preparative 2-D electrophoresis
Whole plasma (20.0 ml) was first dialyzed (2 hours, Mr cut off 10,000) against
distilled water. Following dialysis, urea (21 g, final concentration 7M), CHAPS (1.0
g, final concentration 2% w/v) and DTE (0.232 g, final concentration 30 mM), were
added. After stirring for 15 minutes, carrier ampholytes [Bio-Rad Bio-Lytes
® ; pH
range 3-10 (2.5 ml) and pH range 5-7 (0.5 ml)] were added and the volume was
brought to 50 ml with distilled water.
Preparative Isoelectric Focusing
The sample (50 ml) containing 1.2 grams of total protein, was loaded into the
Rotofor cell for initial fractionation in a wide-range pH gradient (pH 3-10). Constant
power (10 W) was applied for 5 hours with the system cooled to 4 ÞC. Runs were
terminated when the voltage had stabilized (1500 V) for about 30 minutes. 20
Rotofor fractions were collected. Selected fractions were analyzed by 2D-PAGE.
Rotofor fraction 5 (pH 4.3) was substantially free of the bulk plasma proteins,
albumin and immunoglobulins, and highly enriched for Apo J and the unknown
protein. This step provided approximately 500-fold purification of the protein of
interest (Figure 2a).
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