Bio-Rad Rotofor® and Mini Rotofor Cells User Manual
Page 47
Elution buffer:
Tris-Glycine-SDS (25 mM-192mM-0.1%)
Sample buffer A:
10% SDS + 2.32% DTE
Sample buffer B:
1% g DTE + 4% CHAPS + 9 M urea + 5% ampholytes
pH 9-11
Sample:
Rotofor Fraction 11 was dialyzed and freeze dried then
dissolved in 100 microliters of sample buffer A. Then
1900 microliters of sample buffer B was added.
Elution rate:
0.5 ml/min
Power:
start: 50 mA 177 V 8 W
finish: 50 mA 301 V 12 W
Fraction Collection and Analysis
The elution chamber outlet of the Model 491 Prep cell was connected to a
fraction collector (Bio-Rad Econo System) and 80 5-ml fractions were collected.
Fraction number one was the first fraction containing visible amounts of the
bromophenol blue marker dye. In order to locate the fractions containing Apo J
and the 49 kd uncharacterized protein, 30 microliters from every fifth fraction were
analyzed by SDS-PAGE. (Figure 3a). Once the elution positions of the Apo J and
49kd protein were determined, 30 microliters of every fraction near the peak of
the eluted proteins of interest were analyzed by SDS-PAGE. (Figure 3b).
Fig. 3. Analysis of protein fractions eluted from the Prep cell. 3a) Aliquots from every fifth Prep cell
fraction were analyzed on silver stained. SDS-PAGE gels. The elution position of Apo J was fraction 60.
and the 49kd protein eluted in fraction 70. (3b). Every Prep cell fraction near the peak of eluted Apo J
and the 49kd protein was then analyzed. The fractions containing Apo J were 60, 61 and 62. and the
ones containing the 49 kd protein were 71, 72, and 73.
Antibody Production
Rotofor fraction 11 containing Apo J was assayed for protein
6
and frozen at
-20°C until used. Polyclonal antibodies were raised in rabbits against proteins
purified as described above using conventional immunization procedures.
7
Polyclonal antibodies were specific for Apo J and did not cross-react with other
plasma proteins. Using preparative 2-D electrophoresis (combined Rotofor and
43
FRACTIONS
15
20
25
30
35
40
LMW 45
50
55
60
65
70
75
58
59
60
61
62
63
64
65 LMW 66
67
68
69
70
71
72