Biology Reference
In-Depth Information
down to completely resuspend the pellet. Transfer to a 2 mL
low binding tube. Add 1 mL of trypsin buffer to the vial, wash-
ing all the walls. Then transfer to the previous tube. Trypsin
can be stored at −20 °C for 1 month. It is recommended to
make aliquots to avoid melt/freezing cycles that can damage
the enzyme. Aliquots should be stored in low binding tubes.
3. Peptide Solubilization Buffer (PS): 4 % (v/v) acetonitrile,
0.25 % (v/v) formic acid. If properly closed this buffer is stable
for 3 months at room temperature.
3
Methods
3.1 Protein
Extraction
1. Plant and cell samples (10-50 mg of fresh weight) can be
processed using the volumes indicated in this protocol. For
bigger amount of starting material, the proportions and volumes
must be adjusted properly. If not processed immediately after
sampling, store at −80 °C until analysis.
2. Add 300
L of extraction buffer, and pipette up and down
until the sample pellet is completely resuspended. Transfer to a
screw-cap 2 mL tube previously fi lled with 25 mg of quartz
sand and homogenize in a regimill/fastprep for 1 min at maxi-
mum speed. Non pelleted samples can be fi rst transferred to
the screw cap tube, and then add the extraction buffer. Add
100
μ
L of 20 % SDS to the sample tube, mix gently by inver-
sion. Incubate 4-5 min in a shaker at 95 °C. Cool down at
room temperature (
see
Notes 1
and
2
).
3. Centrifuge 5 min at 4 °C in a bench top centrifuge at full speed
to pellet all insoluble materials. Transfer supernatant to a new
2 mL tube (for better yields it is recommended the use low
binding tubes in all of the steps of this protocol).
4. Add at least 4-5 volumes of cold (−20 °C) acetone with 0.5 %
of ß-Mercaptoethanol. Mix it gently by inversion. Keep a mini-
mum of 2 h at −20 °C.
5. Centrifuge 15 min at 5,000 ×
g
and 4 °C. Carefully discard the
supernatant by pipetting it out.
6. Wash the pellet with 1 mL of acetone (0.5 % ß- Mercaptoethanol).
Pipette up and down or use a soft ultrasound bath to disag-
gregate the pellet. Centrifuge 5 min at 5,000 ×
g
and 4 °C to
pellet down the proteins. Use the micropipette for removing
the supernatant, be careful not disturbing the pellet.
7. Wash the pellet with 1.2 mL of acetone. Pipette up and down
or use a soft ultrasound bath to disaggregate the pellet.
Centrifuge 5 min at 5,000 ×
g
and 4 °C to pellet down the
proteins. Use the micropipette for removing the supernatant,
be careful not disturbing the pellet.
μ
