Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 346

molecular mass range from ~50 to 700 kDa. The second-dimension
SDS gel consists of a homogeneous 12.5% separating gel and a 5%
stacking gel. This setup allows the separation of proteins in the
molecular mass range from ~10 to 200 kDa.
The entire experiment was carried out in a Protean II xi system
(Bio-Rad Laboratories GmbH) which has a gel size of 20 × 20 ×
0.075 cm for the fi rst dimension and 20 × 20 × 0.1 cm for the
second dimension.

1. Lyse 1 × 10 8 plastids (see Note 5) in 200 mL of sample buffer

for 10 min. Pellet thylakoid membranes in a microfuge at
7,500 × g at 4°C and remove the supernatant containing all
soluble and peripheral proteins. To be sure that all peripheral
proteins are removed, resuspend the pellet in 200 mL of sample
buffer and centrifuge again. Remove the supernatant.

3.1. Sample
Preparation for Native
Electrophoresis

2. Resuspend the pellet in 70 mL of sample buffer.

3. Transfer 35 mL of the sample to a new tube (sample 1). CyDye

detection requires a lower protein concentration than pigment
fl uorescence detection.

4. Add Cy2 or Cy3 (1 mL of a 400 pmol working solution) to

sample 1, mix, and incubate the sample for 30 min on ice (see
Note 6).

5. Add 1 mL of lysine to sample 1, mix, and incubate the sample

for 10 min on ice.

6. Reunify the labeled sample with the unlabeled one.

7. Mix 30 mL of dodecyl maltoside solution with 60 mL of

digitonin solution and 2 mL of lithium dodecyl sulfate buffer
(see Note 7). Add 15 mL of mixed detergent solution to the
sample and mix gently.

8. Incubate the sample on ice for 10 min to solubilize the protein

complexes.

9. Centrifuge for 10 min at 16,000 × g at 4°C in a microcentri-

fuge to pellet the unsolubilized material. Unsolubilized mate-
rial can affect the subsequent electrophoretic separation of the
protein complexes in a negative way.
10. Transfer the supernatant to a new tube and discard the pellet.

1. Clean glass plates and spacers with denatured ethanol (100%),

assemble the glass plate sandwich, fi x it on the casting stand,
and adjust the assembled casting stand by a spirit level.

3.2. Casting of Native
Gradient PAGE Gels
(First Dimension)

2. Place magnetic stirring rods in both chambers of the gradient

mixer and ensure that all ports are closed.

3.2.1. Separating Gel

3. Prepare the gel solutions stated in Table 1 (see Note 8) directly

in the gradient mixer. Chamber 1 corresponds to the chamber
next to the outlet tube (see Note 9).