Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 243

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4% Stack

20% Gel

Fig. 14.2 Example of membrane rafts isolated as unresolved bands on a 1D SDS-PAGE
gel for in-gel digestion, quantitative labelling and LC-MS/MS analysis. Membrane raft
samples (30 μgin25μL) were loaded into lanes 2, 4, 6, and 8, with prestained molec-
ular weight markers in lane 10 and buffered blanks in lanes 1, 3, 5, 7, and 9.

5. Disconnect the power supply and disassemble the elec-
trophoresis apparatus. Remove the gel cassette from the
tank and rinse with water. Carefully open the cassette,
remove the gel intact, and place into a sterile gel tray ( see
Note 14 ).
6. Add fixing solution and fix the proteins in the gel for
20 min on a rotary platform shaker.
7. Decant the fixing solution, add colloidal coomassie staining
solution, and stain the proteins on a rotary platform shaker
( see Note 15 ).
8. Decant the staining solution from the gel and destain the
gel background for 10 min with destaining solution 1.
9. Decant destaining solution 1 from the gel and continue
to destain the gel with destaining solution 2 for 1 h or
until protein bands are obviously clarified and the gel back-
ground is mostly colourless.
10. Decant destaining solution 2 from the gel and replace with
water. The gel can be stored at 4 ◦ C in the sterile tray
overnight.

3.6. In-GelDigestion

1. Place the gel on a glass pane cleaned with methanol and
place on a light box. Carefully excise protein bands from
the gel with a sharp scalpel ( see Note 16 ). Cut each gel
band into 1-2 mm 3 pieces and place into a 0.5 mL cen-
trifuge tube.
2. Wash the gel pieces twice with 50 mM TEAB, removing
the solutions after washes using a pipette.
3. Dehydrate the gel pieces to small white clumps by washing
twice with acetonitrile, again removing the solutions after
washes using a pipette, then dry the gel pieces fully in a
vacuum centrifuge for 5 min.
4. Prepare a 5 mM TCEP working solution by diluting 10 μ L

of the 500 mM TCEP stock solution with 990 μ Lwater.