Biology Reference
In-Depth Information
11.2.2
In vitro lipid-binding assays with purified recombinant
membrane proteins
11.2.2.1
Binding assays between purified recombinant FIT2 produced
in insect cells and TAG in detergent micelles
11.2.2.1.1
Lipid solubilization
Stock solutions of lipids in chloroform or 1:1 toluene:ethanol that were to be used
in binding assays were dried to completion in Eppendorf tubes under a stream of
nitrogen gas (
5 min). Resuspension was performed by adding a minimal
volume of ethanol (
2% v/v of final reaction volume) and vortexing for 15 s.
Subsequently, the reaction buffer (Buffer C) was added and pipetted up and
down 10 times. Final concentrations of lipids varied from 50 nM to 100
m
M.
Solubilized lipids in 2% v/v ethanol:Buffer C had protein added to them, were
mixed, and aliquotted to Eppendorf tubes containing appropriate amounts of Nickel
or
Strep
-tactin resin.
11.2.2.1.2
Determining protein-lipid interaction by coelution
Purified His
6
-StrepII-tagged recombinant protein in detergent micelles (5
m
g,
150 pmol for FIT2) was added by pipetting to 200
m
l Buffer C containing 50 pmol
(250 nM) [
3
H]-triolein (TAG) dissolved as described in
Section 2.2.1.1
. Pipetting
proceeded until Schlering lines were no longer visible and the mixture was then
added to 30
m
l
Strep
-tactin resin. Binding was permitted to occur over 4 h at room
temperature, after which the reaction was washed twice with 800
m
l of Buffer C by
centrifugation at 16,000
g
for 1 min at room temperature. Using another 800
m
lof
Buffer C, the resin was transferred to a BioSpin column (Bio-Rad) and allowed to
flow through by gravity flow into an Eppendorf tube, which was saved for assaying
3
H radioactivity. The column was transferred to a new tube and elution was begun
with 1 ml of 7.5 mM desthiobiotin in Buffer C. Aliquots of 150
m
l were collected and
assayed for
3
H radioactivity and subjected to immunoblot with anti-His antibody to
detect eluted protein. Coelution was demonstrated by a peak of
3
H radioactivity
coinciding with peak protein densitometry on immunoblot, indicating that FIT2
bound TAG (
Fig. 11.4
).
11.2.2.1.3
Lipid-binding assays with purified recombinant proteins and
neutral lipids
Purified recombinant His
6
-StrepII-tagged proteins solubilized in Fos-choline 13 deter-
gent micelles (3-5
m
g) with desthiobiotin diluted 1000-fold using Amicon MWCO
centrifugation in Buffer C were added to reactions containing [
3
H]-TAG or [
3
H]-
DAG at lipid concentrations of 50 nM to 100
m
M dissolved in 100
m
l of Buffer C.
These mixtures were added to 15-30
m
l Ni-nitrilotriacetate (Ni-NTA) or
Strep-
tactin
slurry (GE Healthcare or IBA, respectively). Reactions proceeded at room temperature
for
4 h, after which beads were washed three times with 800
m
lBufferCby
centrifuging at 16,000
g
for 1 min at room temperature. Beads were then transferred
to 6 ml scintillation vials containing 4 ml scintillation fluid. Resin was transferred to
