Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 351

Fig. 1. Detection of pigment-binding thylakoid membrane complex subunits after native/SDS PAGE. Thylakoid membrane
complexes were labeled by Cy2 and separated by 2D native/SDS PAGE. After electrophoresis, fl uorescent proteins were
detected by a Thyphoon trio scanner scanning for Cy2 (( a ) green ) and Cy5 (( b ) red ) Scanned images of fl uorescent proteins
were overlayed for difference analysis (( c ) green / red / yellow ) .
6. Disassemble the buffer chamber assembly. Clean the glass plate
sandwich with distilled water and scan with a fl uorescence scan-
ner for CyDye and pigment fl uorescence. Scanning for Cy5
fl uorescence is used to image pigment fl uorescence (see Fig. 1 ).
7. After scanning, gels can be used for Coomassie/silver staining
or blotting.
4. Notes
1. Digitonin is dissolved by heating up the solution to 100°C.
After heating, digitonin can immediately be mixed with the
other detergents.
2. If larger amounts of buffers are prepared, it is recommended
to store just a small amount at 4°C and the rest at −20°C.
3. Before use, warm the solution up in warm water, but do not
boil it. Sodium dodecyl sulfate precipitates at 4°C.
4. In general, this protocol can also be used to compare thylakoid
membrane complexes from, e.g., mutant and wild-type plants.
If different thylakoid samples are compared, the amount of
proteins must at least be halved. For the comparison of
two samples, samples are mixed together after labeling and
solubilization and are subsequently separated together by
native PAGE. As the composition of the gel and the anode
buffer correspond to the gel and buffer used in blue native PAGE,
this protocol can also be used to perform blue native PAGE if
the cathode buffer is replaced by the blue native cathode
buffer.
5. Plastids can be counted in an Abbe-Zeiss counting cell chamber.
Instead of counting, the protein amount can be calculated via