Separation of the Plant Golgi Apparatus and Endoplasmic Reticulum by Free-Flow Electrophoresis - Plant Proteomics: Methods and Protocols - page 511

Count e r Flow: FFE Buffer 1

To 96-well collection
plates

96 sampl e outlet
tubes

Deflected
sample stream

Electric field

Electrodes

FFE Buffer 1:
laminar flow in
separation chamber

Electrode circuits
buffer: FFE Buffer 3

Electrode stabilisation
buffer: FFE Buffer 2

FFE Buffer 2

Separation chamber: FFE Buffer 1

Sample inlet tube

Fig. 1 Schematic diagram of zonal electrophoresis-free fl ow electrophoresis
(ZE-FFE) setup showing the laminar fl ow direction in the chamber and progres-
sive defl ection of the sample in the electric fi eld. In this mode the chamber uses
a 0.5 mm spacer and the apparatus is placed horizontally during separation. The
electrodes and FFE buffer 3 are separated from the chamber by membranes and
fi lter papers

Resuspend in 20-80 L of the 10 mM Tris-HCl (pH 7.5),

depending on the pellet size ( see Note 21 ). A fi nal protein
concentration of 0.5-1.0

μ

μ g/ μ L is ideal. Store resuspended

pellets at −20 °C.

1. Digest at least 10 g of protein overnight (37 °C) at a 1:10

trypsin:protein ratio in 40 % (v/v) methanol.
2. Remove methanol in a SpeedVac concentrator until about
1

μ 3.4 Tryptic Digestion

of Samples and
Analysis by Mass
Spectrometry

μ L of sample remains, and then dilute into 25 μ L of the

ACN2 solution.
3. Clean and concentrate samples in Ultra-micro SpinColumns
(10-25 μ L capacity) after initially hydrating the matrix with

H 2 O (75 μ L) for 10 min and centrifuge (1,000 × g , 2 min) as