Biology Reference
In-Depth Information
22.1
DETERGENT-INSOLUBLE GLYCOSPHINGOLIPID (DIG)
MICRODOMAIN ISOLATION
The following protocol is designed for the isolation of detergent-insoluble glyco-
sphingolipid microdomains from cellular membranes, in particular T lymphocytes.
This protocol is modified from methods described previously (
Brown & Rose, 1992;
Field, Holowka, & Baird, 1995; Sargiacomo, Sudol, Tang, & Lisanti, 1993; Xavier
et al., 1998; Darlington et al., 2002
).
22.1.1
Materials
22.1.1.1
Reagents
MES buffer, pH 6.5
NaCl
Triton X-100
EDTA
Na
3
VO
4
(prepared fresh)
PMSF (phenylmethylsulfonyl fluoride)
Aprotinin
Sucrose
Distilled deionized water (ddH
2
O)
22.1.1.2
Equipment
Multi-Purpose Rotator (Scientific Industries Inc.)
Dounce homogenizer with loose-fitting plunger (Wheaton)
Pasteur pipettes
Ultracentrifuge (e.g., Optima XL-90 with SW40 rotor, Beckman)
22.1.1.3
Buffers and sucrose
DIG lysis buffer
(25 mM MES pH 6.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM
EDTA, 1 mM fresh Na
3
VO
4
, 1 mMPMSF, 10
g/mL aprotinin in ice-cold ddH
2
O)
m
FOR 30 ML
750
L of 1 M MES buffer, pH 6.5
m
1500
L of 3 M NaCl
m
1500
L of 10% Triton X-100
m
60
L of 0.5 M EDTA, pH 8.0
m
300
L of fresh 0.1 M Na
3
VO
4
m
300
m
L of 100 mM PMSF
L of 10 mg/mL aprotinin
25.56 mL of ddH
2
O
FOR 50 ML
1250
30
m
L of 1 M MES buffer, pH 6.5
m
2500
L of 3 M NaCl
m
