Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 352

chlorophyll or protein determination; 1×10 8 chloroplasts
correspond approximately to 50 mg of chlorophyll or 400 mg
of membrane proteins.
6. If pigment fl uorescence detection is used via the Cy5 channel,
only Cy2 or Cy3 can be used for protein labeling.
7. The rest of the detergent mix solution can be stored by −20°C
and reused. The detergent mix is soluble at room temperature.
8. The simplest way to transfer the glycerol in the gradient mixer
is to pipette it with a cut 1-mL pipette tip.
9. It is also possible to cast the gradient gel from the bottom of
the gel. In that case, a peristaltic pump is needed and the risk
of air bubbles in the gel during the casting process is higher.
10. Polymerization of the separating gel is completed when a layer
of water is formed between the upper edge of the gel and the
isobutanol layer.
11. After wrapping the gel sandwich in wet tissues and a plastic
bag, it can be stored up to 1 week at 4°C. In case of SDS gels,
the gel should be stored without the stacking gel because of
the different pH of the separating and stacking gel.
12. You can add 2 mL of 80% glycerol to facilitate the application
of the samples.
13. Beside the use of microsyringes, it is also possible to use gel
loader tips to underlay the samples into the wells. As gel loader
tips are much more fl exible than a microsyringe and end in a
capillary tip, it is easier to apply the samples between the glass
plates.
14. The addition of bromphenol blue facilitates tracing of the
running front during electrophoresis.
15. The electrophoretic run can also be performed in a cold room
at 4°C if no thermostatic cooler is available. It is advisable to
cool the gel during electrophoresis to keep the complexes
intact, maintain their structure, and protect them against
proteolysis.
16. The second-dimension gel must be thicker than the fi rst-
dimension gel for inserting the fi rst-dimension gel strip between
the glass plates. Therefore, 0.75-mm spacers are used for the
fi rst dimension, and 1-mm spacers are used for the second
dimension in this protocol.
17. The addition of urea to the gel aids to keep the secondary
structure of the membrane proteins denatured during SDS
PAGE electrophoresis.
18. The experiment can be interrupted after cutting the fi rst-
dimension gel lanes. Lanes can be stored at −20°C in a plastic
folder for some weeks before processed further.