Biology Reference
In-Depth Information
8. 5 % (v/v) trifl uoroacetic acid (TFA)/50 % (v/v) acetonitrile:
Mix 450
μ
L of water and 500
μ
L of acetonitrile in a 1.5 mL
microtube. Quickly add 50
μ
L of TFA into the solution and
mix well (
see
Note 11
).
9. 0.1 % (v/v) TFA: Quickly add 1
μ
L of TFA into 999
μ
L of
water and mix well (
see
Note 11
).
All of these processes must be carefully performed at a clean bench
with gloves and a clean lab coat throughout to avoid contamination
by keratin, dust, and other exogenous proteinaceous materials.
2.4 In-Solution
Tryptic Digestion
1. MPEX PTS reagents kit (GL science Inc., Tokyo, Japan):
Make solution B, DTT solution, IAA solution, and trypsin
solution according to the manufacturer's instruction manual.
Only solution B should be stored at 4 °C. Prepare fresh DTT
solution, IAA solution, and trypsin solution immediately
before use.
2. 5 % (v/v) acetonitrile/0.1 % (v/v) TFA: Add 50
μ
L of aceto-
nitrile and 1
μ
L of TFA into 949
μ
L of water and mix well (
see
Note 11
).
3. Pierce BCA protein assay kit (Thermo Fisher Scientifi c, MA,
USA): Store at room temperature.
2.5 Peptide
Purifi cation
Components
1. SPE C-TIP T-300 (Nikkyo Technos Co., Ltd., Tokyo, Japan).
2. 1.5 mL microtubes: Make a hole of 3 mm in diameter on the
cap with a soldering iron. Prepare two tubes per sample.
3. Solution A: Add 800
μ
L of acetonitrile and 5
μ
L of TFA into
L of water and mix well (
see
Note 11
).
4. Solution B: Add 40
195
μ
μ
L of acetonitrile and 5
μ
L of TFA into
L of water and mix well (
see
Note 11
).
5. 0.1 % (v/v) TFA: Quickly add 1
955
μ
μ
L of TFA into 999
μ
L of
water and mix well (
see
Note 11
).
3
Methods
Wear gloves and a clean lab coat throughout the processes to avoid
contamination by keratin, dust, and other exogenous protein-
aceous materials. It is preferable to use low-protein-absorption
microtubes at all stages.
3.1 Plasma
Membrane Purifi cation
Perform all steps on crushed ice (unless indicated otherwise).
Schematic outline of the procedure is described in Fig.
1
.
1. Cut out the aerial parts of oat seedlings and weigh the samples
(10-70 g in fresh weight is suitable for the plasma membrane
purifi cation). Put the harvested plants on a plastic container
