Biology Reference
In-Depth Information
MW
1
F
1
W
2
F
2
1236
1048
720
480
242
146
66
20
Fig.
1
Separation of thylakoid membrane protein complexes by native LDS-
PAGE. Thylakoid membranes corresponding to 8
L
detergent mixture 1 containing 20 mM Digitonin, 0.073 mM LDS, 20 % (v/v)
Glycerol, 0.01 % (w/v) Ponceau S, 15 mM Tricine, 80 mM Bis-Tris (W
1
, F
1
) and
detergent mixture 2, 9 mM Digitonin, 9 mM
n
-dodecyl-
μ
g Chl were solubilized in 20
μ
-
D
-maltoside, 0.073 mM
LDS, 20 % (v/v) Glycerol, 0.01 % (w/v) Ponceau S, 15 mM Tricine, 80 mM Bis-Tris
(W
2
, F
2
). Non-solubilized material was separated by centrifugation (14,000 ×
g
,
10 min, 4 °C) and discarded. Supernatant containing the solubilized protein com-
plexes was directly loaded onto native LDS-PAGE. Chl-protein complexes were
detected by white light scanning (W
1
, W
2
) and fl uorescence excitation scanning
(F
1
, F
2
) (Ex 680 nm and Em >694 nm). Standard proteins (M, kD) were stained
with colloidal Coomassie G250
β
10. Centrifuge 5 min at 6,000 ×
g
.
11. Supernatant = chloroplast stroma. Purify stroma from residual
thylakoids by centrifugation at 12,000 ×
g
for 30 min.
12. Pellet = chloroplast thylakoid membrane.
13. Gently resuspend thylakoid membrane pellets from both tubes
in 5 mL of wash buffer with a paintbrush and collect in one
tube.
14. Collect thylakoids by centrifugation for 5 min at 5,900 ×
g
.
15. Discard the supernatant and keep the thylakoid pellet.
Resuspend thylakoids in 3 mL wash buffer using paintbrush.
16. Transfer into glass homogenizer and lever piston three times.
17. Transfer into 5 mL plastic tube
18. Clean homogenizer with 2 mL of wash buffer and homoge-
nize 2× and collect in 5 mL tube.
