Biology Reference
In-Depth Information
Microdomains
Add 900
L of DIG lysis buffer to one tube, vortex, then transfer mixture to the
other tube, and vortex.
Centrifuge the beads coated with antibody (i.e., maximum speed for 3 s). Discard
the supernatant and wash the antibody-coated beads once with 500
m
LofDIG
m
lysis buffer. Centrifuge again and discard the supernatant.
Add the DIG fraction to the antibody-coated beads and then rotate gently at 4
C
for 2 h or overnight.
Wash three times with 1 mL of DIG lysis buffer and centrifuge at maximum
speed for 3 s each time.
Add 2
sample buffer, boil at 95
C for 5 min, and then proceed with Western
blotting.
Note
: Soluble fractions should be immunoprecipitated directly.
22.2
PLASMA MEMBRANE ISOLATION
This protocol is designed to obtain highly purified plasma membranes for biochem-
ical studies and also to produce an internal membrane fraction. This protocol has
been adapted from
Chaney and Jacobson (1983)
and
Spector, Goldman, and
Leinwand (1998)
.
22.2.1
Materials
22.2.1.1
Reagents
MES buffer
NaCl
Sorbitol
Polymethacrylic acid (PAA)
Imidazole
Aprotinin
Leupeptin
PMSF
Na
3
VO
4
Ludox
Histodenz
Sodium dodecyl sulfate (SDS)
b
-Mercaptoethanol
Tris
Glycerol
Bromophenol blue
ddH
2
O
22.2.1.2
Buffers and solutions
PMCB (plasma membrane coating buffer)
20 mM MES
