Biology Reference
In-Depth Information
3
Methods
Carry out all procedures at room temperature unless otherwise
specifi ed.
3.1 Sample
Preparation
1. Filter cells (5 g) through a Büchner funnel glass fi lter and wash
them twice with 250 mL of water. Freeze immediately the cells
in liquid nitrogen before storage at −85 °C until use.
2. Grind cells in a blender or in a mortar (precooled with liquid
N
2
) and dissolve the resulting powder in 30 mL of preheated
(45 °C) extraction buffer (HENS) made of 150 mM Hepes
buffer pH 7.7, 5 mM EDTA, 0.5 mM neocuproine, and 1 %
SDS (preheating is used to avoid SDS precipitation). Filter
quickly the solution through a 38
m sieve using a vacuum
pump and a funnel. Split the fi ltrate (25 mL) in 5 × 5 mL in
50 mL centrifuge tubes and dilute the fi ltrate (ratio 1:9) in
cold acetone (−20 °C) (
see
Note 7
). Keep at −20 °C for at least
30 min to precipitate proteins. Spin the 50 mL centrifuge
tubes at 6,000 ×
g
for 15 min and collect the pellet. Allow pel-
let to dry under a hood for 15 min. Resuspend proteins in
5 mL of HENS buffer and quantify the protein concentration
using the 2D Quant kit assay. Take out 2 mg of proteins and
keep a protein concentration below 0.8 mg/mL (dilute your
sample in HENS buffer if necessary).
μ
1. Incubate the sample (2 mg of proteins) in a 1.5 mL centrifuge
tube with 100 mM MMTS (20 min, 50 °C) to block free thiols
(Fig.
1
). Do not exceed 0.8 mg/mL of protein to keep the
blocking step as effi cient as possible. After the MMTS blocking
step, dilute your sample with HENU buffer to keep your SDS
concentration below 0.1 % (
see
Note 8
). MMTS excess is
removed by fi ltration using fi lter devices. To condition the fi lter
devices (Amicon Ultra-15) add 15 mL of water and spin for
10 min at 5,000 ×
g
(fi xed angle). Repeat this step twice and
replace water by the HENU buffer for another step condition-
ing. Load the sample to the fi lter device and spin for 10 min at
5,000 ×
g
. Wash the sample three times (ratio 1:100) with
HENU buffer on fi lter devices and a fourth time with HEN to
dilute the urea at the end of the washing process. Make sure that
the solution (wash buffer and sample) are well homogenized by
pipetting up and down the solution in the fi lter device before
centrifugation. Transfer the sample to a 0.5 mL fi lter device
(Amicon Ultra-0.5) after fi lter device conditioning and add
HEN buffer to the sample to bring the volume up to 500
3.2 BS-ICAT Labeling
2. To reduce nitrosothiols, add 5 mM ascorbic acid and 1 mM
CuCl
2
(an enhancer of the reaction) and incubate in the fi lter
device for 1 h at RT (Fig.
1
;
see
Note 9
).
μ
L.
