Biology Reference
In-Depth Information
13.1
MATERIALS
INSTRUMENTS
Ultracentrifuge
Luminescent image analyzer
Luminescence plate reader (with emission filters 370-450 and 500-530 nm), we
used a FLUOstar OPTIMA (BMG Labtech)
Fluorescence plate reader (with excitation at 488 nm and emission at 515 nm), we
used a FLIPR (Molecular Devices)
VECTORS
Mammalian expression vector, for example, pcDNA3 (Invitrogen), pcDNA5/
FRT/TO (Invitrogen), and pEAK10 (Edge Biosystems)
BRET vector, pGFP
2
-MCS-Rluc(h) (PerkinElmer)
CELL LINES
HEK293T cells (we used a modified cell line stably expressing the G
a
protein
chimera G
a
16gust44
)
CELL CULTURE DISHES AND PLATES
10 cm cell culture dishes, coated with poly-
D
-lysine
96-Well plates, white, clear-bottomed (Greiner Bio-One); coatedwith poly-
D
-lysine
96-Well plates, black, clear-bottomed (Greiner Bio-One); coated with poly-
D
-lysine
BUFFERS AND SOLUTIONS
PBS (140 mMNaCl, 10 mMNa
2
HPO
4
, 2.7 mMKCl, and1.76 mMKH
2
PO
4
,pH7.4)
C1 buffer (130 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl
2
, and 1 mM
glucose, pH 7.4)
CoIP buffer (120 mM NaCl; 50 mM Tris, pH 8.0; 1 mM EDTA, pH 8.0; 0.5%
(v/v) IGEPAL; 1 mM PMSF; 2
m
g/ml leupeptin; 2
m
g/ml pepstatin A; and 10
m
g/
ml aprotinin)
2
Laemmli buffer (125 mM Tris, pH 6.8; 4% (w/v) SDS; 20% (v/v) glycerol;
and 0.04% (w/v) bromophenol blue) without DTT or mercaptoethanol!
TBS (10 mM Tris, pH 8.0, and 150 mM NaCl)
TBST (10 mM Tris, pH 8.0; 150 mM NaCl; and 0.2% (v/v) Tween)
ANTIBODIES
Rabbit anti-FLAG
®
antibody (Sigma-Aldrich, catalog #F7425)
Rabbit anti-GFP antibody (Abcam, catalog #ab6556)
Donkey anti-rabbit IgG, coupled to horseradish peroxidase (Amersham/GE
Healthcare)
EZview
TM
Red anti-FLAG
®
M2 affinity gel (Sigma-Aldrich, catalog #F2426)
