Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 48)

Prep cell) we were able to obtain a pure preparation of Apo J, free of cross-
contamination, despite the presence of a series of presumably glycosylation-induced
isomers. Figure 1.
Sequence Analysis
Prep cell fractions 71, 72 and 73 containing the 49 kd unknown protein, were
pooled and concentrated to 500 microliters by freeze drying. The final concentrations
of components in the 500 microliter sample was: Tris (200 mM) - glycine (1.6 M) -
SDS (0.8%). The sample was then reduced with DTT (2.0 µM, 2 hours at 37 ÞC) and
carboxymethylated with iodoacetic acid (ICH
2
COOH, 5 µM, pH 8) for 30 minutes in
the dark. Following dialysis against water for 48 hours, the sample was again freeze
dried, and the SDS extracted.
8 The protein was then digested with TPCK-trypsin in
4 molar urea, pH 8.0. Prior to sequencing, peptides were separated with a Microbore
C8 HPLC column (1x100mm) with a 0.1% TFA/Acetonitrile system.
9 Sequence
analysis was done on an ABI 473 A sequenator. We have found no sequences similar
to those of the 49 kd protein in data base searches.
13.3 Results
This report describes a rapid electrophoretic procedure for purification of proteins
from crude extracts in concentrations where comprehensive sequence analysis and
antibody production are feasible. Apo J and the 49kd uncharacterized protein were
obtained in a highly purified state. The preparative 2-D procedure typically yields
from 20-40 micrograms of the proteins.
The advantages of the primary fractionation step (liquid phase IEF) cannot be
over-emphasized, notably with respect to the fractionation of plasma proteins.
Here, high plasma concentrations of certain proteins, such as albumen, alpha-1-
antitrypsin, immunoglubulins or transferrin limit the volume of plasma that can be
processed. Pre-fractionation of plasma with the Rotofor confines these proteins to
their respective pl ranges. It is then possible to undertake a sequential, detailed
analysis of the different Rotofor fractions. Each fraction represents a defined,
restricted pl interval, containing an adequate quantity of protein for the preparative
PAGE purification step.
In conclusion, this procedure can be considered a viable means of obtaining
highly purified preparations of plasma proteins, even those present in low
concentrations. Yields are such that comprehensive sequence data can be generated
on amino-terminally blocked proteins and antibody production is feasible. The
procedure offers great potential as a firstline protein purification procedure,
whether applied to plasma or other biological samples.
References
1. O’Farrel 1975, J. Biol. Chem., 250, p.4007-4021
2. Andersonn and Anderson 1984; Clin. Chem., 30, p. 1898-1905
3. Hochstrasser et al. 1988a, Anal. Biochem., 173, p.424-435
4. Bauws G. et al., Proc. Natl. Acad. Sci. USA, 1989, 86, p. 7701-7705
5. Hochstrasser et al. 1990, Applied and Theoretical Electrophoresis, 1, p.265-275
6. Lowry et al. 1951, J. Biol. Chem., 193, p. 265-275
7. James R.W. et al, Arteriosclerosis and Thrombosis, 1991 May/June, 11, No.3, p. 645-652
8. Konigsberg, W.H., and Henderson L., 1990, Meth. Enzym., 91, p. 254
9. Hughes et al., Biochem. J, 1990, 271, p. 641-647
*Contributed by Jean Charles Sanchez, Nicole Paquet, Graham Hughes and Denis Hochstrasser Medical
Biochemistry Dept., Geneva University.
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