Biology Reference
In-Depth Information
1. 80-130 g maize roots are harvested within 30 min and
homogenized with 200 mL homogenization buffer
(Subheading
2.1
), supplemented with 1 mM DTT and 1 %
insoluble PVPP, using a warring blender (3 × 15 s with a break
of 20 s between each homogenizing step). The crude extract is
then fi ltered through a nylon net (125
m mesh; Hydrbios,
Germany) and 2 mL 100 mM PMSF are added.
2. Intact cell organelles and cell walls (pellet) are separated from
the soluble protein fraction and membrane protein fraction
(supernatant) by centrifugation at 9,000 ×
g
for 10 min.
3. The supernatant containing soluble protein fraction and micro-
somes are separated at 50,000 ×
g
for 30 min.
4. The achieved microsomal pellet is resuspended in phase buffer
(Subheading
2.3
), but the supernatant (soluble proteins) is
also kept. Microsomes and soluble fractions are stored below
−70 °C until further use.
μ
3.2
Soluble Proteins
1. All soluble proteins (achieved in Subheading
3.1
) are precipi-
tated with ammonium sulfate, which is added slowly, under
stirring, to the solution (
see
Note 5
) until the concentration
reaches 90 % (662 g/L) (
see
Note 6
).
2. The precipitation is accomplished over night at 4 °C under
continues stirring.
3. Centrifuge the solution at 50,000 ×
g
for 30 min.
4. Resuspend the pellet in 25-50 mL (
see
Note 7
) resuspension
medium S (Subheading
2.2
) and desalt 500
L sample using
centrifugal fi lter units cut off 10,000 MWCO (Millipore,
Germany). After desalting and concentration determine the
protein concentration (
see
Note 8
). The fi nal protein concen-
tration should be adjusted to 1-5 mg/mL by fi lling with resus-
pension medium S. Store the sample till use below −70 °C.
μ
3.3 Plasma
Membrane Preparation
by Aqueous Polymer
Two-Phase
Partitioning
1. Plasma membranes are purifi ed from microsomal fractions
(about 80 mg protein) by six steps of aqueous polymer two-
phase partitioning using a 36 g phase system consisting of 6.5 %
(w/w) dextran T500, 6.5 % (w/w) PEG 3350, 250 mM sucrose,
5 mM KCl, 5 mM phosphate buffer, pH 7.8 (Fig.
1
) (
see
Note
9
).
2. 9 g of microsomal fraction (
see
Subheading
3.1
,
step 4
; keep a
part of the microsomal sample for the later analysis) are added
to a 27 g phase mixture. The phase system is then mixed thor-
oughly by 20-30 inversions of the tube and centrifuged at
1,000 ×
g
for 5 min in a swing-out rotor.
3. The resulting upper phase is transferred to the next lower
phase, resulting in a 36 g phase system (
see
Note 10
), mixed and
centrifuged as explained in the previous step. This procedure is
