Separation of Membrane Protein Complexes by Native LDS-PAGE - Plant Proteomics: Methods and Protocols - page 642

3.6 Loading and
Running of Native LDS
Electrophoresis

1. Label the position of the wells on the outer glass plate with a
felt pen and remove the comb carefully from the native gel
( see Note 13 ).
2. Assemble glass sandwich within electrophoretic apparatus.
3. Fill in cathode buffer into the upper buffer chamber.
4. Rinse the wells with cathode buffer ( see Note 14 ).
5. Underlay the samples into the wells using a microsyringe or
disposable pipette tips ( see Note 15 ).
6. Fill anode buffer in the lower buffer chamber until electrode is
immersed.
7. Connect the tubes of the thermostatic circulator to the buffer
chamber. Start the thermostatic circulator. Perform electro-
phoretic run at 4 °C ( see Note 16 ).
8. Complete assembly of electrophoresis unit and connect to
power supply.
9. Set the power supply to limit voltage. Use 150 V constant. Set
mA and W to maximal values. Run overnight for highest reso-
lution. Apply power to electrophoretic set.
10. Stop electrophoresis when the front composed of Ponceau S/
has reached the bottom of the separating gel ( see Note 17 ).
11. Stop the thermostatic cooler and disassemble the buffer cham-
ber assembly.
12. After electrophoresis, use glass plate sandwich without disas-
sembly for detection of binding protein complexes by scanning
with white light or fl uorescence scanners (Fig. 1 W 1/2 and
F 1/2 ).
13. Scan native gels for fl uorescence emission by laser excitation at
680 nm and emission wavelength 694 nm using the Odyssey ®
infared imaging system (LI-Cor Biosciences) or use alternative
scanner like Thyphoon Trio (GE Healthcare) by laser excita-
tion at 633 nm and emission of >670 nm using a BP30 emis-
sion fi lter ( see Note 18 ).
14. For further analysis of protein complexes like absorbance or
mass spectroscopy measurements open sandwich and cut gel
bands or stain proteins before further processing of sample of
interest.
15. This robust high-resolution separation protocol for membrane
protein complexes is free of Coomassie and can be operated at
0 °C. Gels are fully transparent. Fluorescent labeling before
and fl uorescent readout after the separation has shown excel-
lent results throughout the separation range (Fig. 1 ). Kinetic
studies during biogenesis of membranes and assembly of the
proteins can be combined with enzyme assays, gel-blot identi-
fi cations and mass spectrometry of proteins directly to identify
the native complexes within the gel.