Biology Reference
In-Depth Information
5. Repeat
step 4
. At this step the supernatant must be almost
transparent or with a very light blue color, if the blue color is
intense, repeat this step (
see
Note 7
).
6. Dehydrate gel pieces with 300
L 100 % Acetonitrile (AcN)
incubate 5 min at room temp in a shaker. Discard supernatant.
At this point gel pieces should be very reduced in volume,
hard, and completely white, having a “plastic” look. If they are
still translucent or soft (you can use the pipette tip to test it)
repeat this step.
7. Dry out in a SpeedVac for 5 min.
8. Add 50
μ
L of Trypsin Solution to each tube and incubate
15 min at 37 °C. If needed, add more trypsin solution until the
gel pieces are completely rehydrated. Wait another 15 min.
Completely cover the gel pieces with trypsin buffer.
9. Incubate 14-16 h at 37 °C. Incubation in an oven is recom-
mended, if a thermal block is used, cover it with aluminum foil
to maintain the temperature also in the caps of the tubes.
Shaking during the digestion is not advised (
see
Note 8
).
μ
1. Add 150
L of 50 % ACN/1 % formic acid (FA) to each tube
with gel pieces and incubate 5 min at room temperature. After
incubation sonicate 3 min in a low intensity ultrasound bath.
2. Spin the tubes and transfer the supernatant to a new tube (“A”).
3. Repeat 1. Transfer the supernatant to tube A.
4. Add 100
μ
3.4.2 Peptide Extraction
L of 90 % ACN/1 % formic acid (FA) and incubate
for 5 min at room temperature. Transfer the supernatant to the
A tube. The gel pieces should look in the same way than in
step 6
of protein digestion. If not, repeat this step once more.
5. Dryout in SpeedVac. Keep at −20 °C or proceed with desalting.
For long term storage −80 °C is recommended.
μ
1. Add 75
μ
L of PS Buffer to peptide pellets. Incubate for 10 min
3.4.3 Peptide Desalting
on ice.
2. Resuspend the pellet by pipetting and sonicate 3 min in a low
intensity ultrasound bath. Incubate 5 min at room temperature
and then keep on ice.
3. Use C18 stage tips or 96-well plates for peptide desalting.
Independently of the chosen support the protocol remains
unchanged (only volumes should be adjusted). All of the steps
are performed at room temperature. We routinely use 96-well
plates (Spec 96-Well C18, Agilent) as follows:
(a) Plate activation and washing: Add 700
μ
L MeOH to each
well (repeat once) (
see
Note 9
).
(b) Plate washing and equilibration: Add 700
μ
L
dd
H
2
O to
each well (repeat once).
