3. Leave 20 min on ice. Vortex and do a quick spin to bring all
samples down.
4. Heat at 37
C for 30 min (see Note 18). Vortex and do a quick
spin to bring all samples down.
5. Heat at 100
C for 20 min. Vortex and do a quick spin to bring
all samples down.
6. Leave at 60
C for 2 h. Vortex and do a quick spin to bring all
samples down.
7. Sonicate for 5 min and do a quick spin to bring all
samples down.
8. The lysates are now ready for (1) RCDC protein assay if the
protein concentration needs to be measured (see Note 19);
(2) add Laemmli buffer to run samples in a Bis-Tris gel if a
set number of cuts were obtained (see Note 20); or (3) directly
proceed to step 1 of Subheading 3.8.
3.7 Sample
Preparation for Mass
Spectrometry Using
Limited Gel
1. Use a 7.5% Mini-Protean TGX gel or, alternatively, you can
polymerize an 8% acrylamide Bis-Tris gel (see Note 21).
2. Calculate the protein concentration for each sample (for example, for the orbitrap LC-MS/MS, we ran 2 and 5 μg of protein)
or load a predetermined number of cuts (see Note 20).
3. Add the corresponding volume of 5Â Laemmli buffer containing 150 mM DTT, vortex, and spin the samples.
4. Boil (100
C) the samples for 5 min.
5. When the samples are cool, vortex and spin again.
6. Load the samples on the acrylamide gel.
7. Run the gel at 30 mA, or about 5–10 min, down to ~1 cm
inside the gel (enough to visualize the MW marker ladder
separation) (see Note 22).
8. Stop running the gel and fix it for 60 min in the fixing solution
(water:methanol:acetic acid ¼ 40:40:8).
9. Inside a biosafety cabinet, excise each lane carefully from the gel
(see Note 23).
10. Put the sliced gel piece in a 1.5-mL centrifuge tube filled with
1% acetic acid in molecular biology grade water. The samples
can be stored in this solution at 4
C until analyzed by MS.
11. Standard MS sample preparation should be performed (see
Note 24).
12. Briefly, for in-gel digestion, dice the samples into 1 mm  1 mm
squares, rinse multiple times with 50 mM ammonium bicarbonate, and reduce with 5 mM DTT and 50 mM ammonium
bicarbonate at 55
C for 30 min.
38
Ana Gordon and Karine Gousset
samples down.
4. Heat at 37
C for 30 min (see Note 18). Vortex and do a quick
spin to bring all samples down.
5. Heat at 100
C for 20 min. Vortex and do a quick spin to bring
all samples down.
6. Leave at 60
C for 2 h. Vortex and do a quick spin to bring all
samples down.
7. Sonicate for 5 min and do a quick spin to bring all
samples down.
8. The lysates are now ready for (1) RCDC protein assay if the
protein concentration needs to be measured (see Note 19);
(2) add Laemmli buffer to run samples in a Bis-Tris gel if a
set number of cuts were obtained (see Note 20); or (3) directly
proceed to step 1 of Subheading 3.8.
3.7 Sample
Preparation for Mass
Spectrometry Using
Limited Gel
1. Use a 7.5% Mini-Protean TGX gel or, alternatively, you can
polymerize an 8% acrylamide Bis-Tris gel (see Note 21).
2. Calculate the protein concentration for each sample (for example, for the orbitrap LC-MS/MS, we ran 2 and 5 μg of protein)
or load a predetermined number of cuts (see Note 20).
3. Add the corresponding volume of 5Â Laemmli buffer containing 150 mM DTT, vortex, and spin the samples.
4. Boil (100
C) the samples for 5 min.
5. When the samples are cool, vortex and spin again.
6. Load the samples on the acrylamide gel.
7. Run the gel at 30 mA, or about 5–10 min, down to ~1 cm
inside the gel (enough to visualize the MW marker ladder
separation) (see Note 22).
8. Stop running the gel and fix it for 60 min in the fixing solution
(water:methanol:acetic acid ¼ 40:40:8).
9. Inside a biosafety cabinet, excise each lane carefully from the gel
(see Note 23).
10. Put the sliced gel piece in a 1.5-mL centrifuge tube filled with
1% acetic acid in molecular biology grade water. The samples
can be stored in this solution at 4
C until analyzed by MS.
11. Standard MS sample preparation should be performed (see
Note 24).
12. Briefly, for in-gel digestion, dice the samples into 1 mm  1 mm
squares, rinse multiple times with 50 mM ammonium bicarbonate, and reduce with 5 mM DTT and 50 mM ammonium
bicarbonate at 55
C for 30 min.
38
Ana Gordon and Karine Gousset