Biology Reference
In-Depth Information
3
Methods
This protocol describes how protein assemblies can be separated by
a native PAGE approach. The method uses the anionic detergent
LDS in the cathode buffer lending the name native LDS-PAGE.
This detergent was selected because of its high solubility at 4 °C.
In general, native PAGE is intended to separate protein complexes
in a state that refl ects the physiologically relevant functional state
of the protein assembly. It is therefore advisable to minimize pro-
tein complex degradation through sample preparation on ice and
electrophoresis at 4 °C. Assemblies of chlorophyll-binding thyla-
koid membrane proteins were found to remain intact in the native
LDS-PAGE approach and were easily detected by white light and
fl uorescence scanning after electrophoresis (Fig.
1
). Separation of
protein complexes by native PAGE is based on a sieving of the
complexes by the acrylamide concentration-dependent pore size of
the gel. The method presented here corresponds to a 3.5-12 %
(v/v) linear gradient separating gel and a 3 % (v/v) stacking gel
(Fig.
1
). This concentration facilitates a separation of molar mass
standards in the range of 20-1,500 kDa (Fig.
1
). Experiments
were carried out in a Hoefer SE400 electrophoresis chamber
(vertical slab unit).
3.1 Arabidopsis
thaliana Sample
Preparation
Arabidopsis thaliana
plants are grown on soil for 3-4 weeks in a
growth chamber with ambient white light of 100
μ
mol/m/s at a
light-dark cycle of 8 h-light-16 h-dark.
1. Homogenize leaves 4× 4 s in 150 mL of ice-cold extraction
buffer.
2. Filter homogenized leaves through one layer of Miracloth and
collect through a funnel in 3× 50 mL conical tubes.
3. Centrifuge for 3 min at 1,800 ×
g
. Collect the pellet and dis-
card the supernatant.
4. Gently resuspend the pellet in 5 mL extraction buffer using a
paintbrush. Wash the brush in 25 mL extraction buffer.
Redistribute into two conical tubes and fi ll to 50 mL with
extraction buffer.
5. Centrifuge for 3 min at 1,800 ×
g
. Collect the pellet and dis-
card the supernatant.
6. Gently resuspend each pellet in 5 mL of lysis buffer with a
paintbrush.
7. Combine resuspended material in one conical tube and fi ll to
50 mL with lysis buffer.
8. Divide sample into two Sorvall HB4 tubes and fi ll to 25 mL
with lysis buffer.
9. Incubate sample 5 min in the dark on ice.
