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

and frozen at

-20°C until used. Polyclonal antibodies were raised in rabbits against proteins
purified as described above using conventional immunization procedures.

Polyclonal antibodies were specific for Apo J and did not cross-react with other
plasma proteins. Using preparative 2-D electrophoresis (combined Rotofor and

FRACTIONS

LMW 45

65 LMW 66