Biology Reference
In-Depth Information
contaminants [
5
] have demonstrated the benefi t of surface charge
purifi cation of organelles with similar density to other subcellular
compartments.
During ZE-FFE, a sample is loaded into a separation chamber
in which separation buffers are moving under laminar fl ow. A volt-
age is applied across the separation chamber, perpendicular to
buffer fl ow, whilst opposing buffer fl ow at the top of the chamber
directs the separated sample into 96 collection tubes [
6
]. Extensive
parameter optimization is possible when using FFE; multiple varia-
tions on the buffer system described here exist in the literature and
chamber height (0.5 mm for standard ZE-FFE) can be adjusted by
spacers of varying thickness. The diameter of pump tubing deliver-
ing buffers can, within certain constraints, be altered. Sample and
carrier buffer fl ow rates are adjustable, as is the voltage. The fl exi-
bility and adaptability inherent in this technique have proved
essential in enhancing the separation of organelles with similar
charge and density such as mitochondria and peroxisomes [
3
,
4
]
and the Golgi/other secretory compartments [
5
]. In the specifi c
case of the endomembrane, although there has been a long history
of separating this system using density gradients [
7
], the limited
degree of resolution achieved has necessitated involved analytical
techniques to adequately tease apart these integrated membrane
systems [
8
].
Here we describe a protocol for preparation of purifi ed plant
Golgi and highly enriched ER samples for proteomic analysis from
an endomembrane-enriched sample using ZE-FFE. We also
describe the initial pre-FFE enrichment and a brief example of
proteomic data processing. These methods were developed using
suspension-cultured Arabidopsis cells; however the same protocol
has been applied to cultured cells of other species and other sources
of plant tissue.
2
Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a sensitivity of 18 M
cm at 25 °C) and
analytical grade reagents. Prepare all reagents at room tempera-
ture. Perform all centrifugation steps at 4 °C. Unless otherwise
stated, prepare all buffers the day before and store at 4 °C.
Ω
1. Temperature-controlled shaking incubator (23 °C, 120 rpm)
with constant light (100
2.1 Arabidopsis
Protoplast Preparation
E).
2. Arabidopsis cell culture medium: 2 % (w/v) sucrose,
α
μ
-naphthaleneacetic acid (0.5 mg/L), kinetin (0.05 mg/L),
1× Murashige and Skoog basal salt mixture [
9
]. Prepare media
