- Phosphoproteome: Sample Preparation 41
- 1 M Triethylammonium bicarbonate (TEAB) (CAS NO. 15715-58-9)/Tris-
HCl (pH = 9.0) - 1.5 mL microcentrofuge tube
Procedure:
I. Prepare 120 mM SDC in H 2 O.
II. Prepare 120 mM SLS in H 2 O.
III. Prepare cell lysis buffer (freshly prepared): 12 mM SDC, 12 mM
SLS, 100 mM TEAB/Tris-HCl, and protease/phosphatase inhibitors
(Table 1).
IV. Cell collection
Method I: Wash cells with PBS and harvest cells in ice-cold lysis
buffer by scrapping. Immediately store samples at− 80 ◦C
until use.Hint: Remove PBS completely before the addition of
lysis buffer. The remaining PBS could dilute the lysis
buffer.Method II: Wash cells with PBS. Harvest cells by trypsinization and
centrifuge at 1,000×g for 5 minutes. Cell pellet can be
stored at− 80 ◦C until use. Resuspend the pellet in ice-cold
lysis buffer before sonication.
V. Lyse cells using sonication with suitable amplitude and time (60%
amplitude and 0.6 cycle for cultured cells using Sartorius LAB-
SONIC M). Keep samples in ice-water bath to avoid the heating of
samples. The lysate should become clear after sonication.Table 1. Recipe for cell lysis buffer.
PTS buffer for cell lysis 1 mL
120 mM SDC 100 μL
120 mM SLS 100 μL
1 M TEAB or Tris-HCl (pH 9.0) 100 μL
Protease inhibitor cocktails 10 μL
Ser/Thr phosphatase inhibitor cocktails 10 μL
Tyr phosphatase inhibitor cocktails 10 μL
MilliQ 670 μL