Biology Reference
In-Depth Information
70
60
50
40
30
20
10
0
0
20
40
60
80
100
pmol [
3
H]-TAG added
FIGURE 11.5
Binding of [
3
H]-TAG to FIT2-His
6
-StrepII is saturable. A dose-response curve of
FIT2-His
6
-StrepII binding to [
3
H]-TAG shows saturable binding kinetics. Data are represented
as mean
SD.
Adapted from
Gross et al. (2011)
.
[
3
H]-TAG were dried down with 30-120 nmol unlabeled TAG and resolubilized/
vortexed in 6
m
l of ethanol. After vortexing, 300
m
l of Buffer C was added by pipet-
ting, 9
m
g of FIT2 (in 9
m
l) was added by gentle pipetting, and 100-103
m
l was added
to each of three tubes containing 15-30
m
l Ni-NTA resin. Reactions were allowed to
proceed at room temperature for 4 h and were then washed three times with 800
m
lof
Buffer C by centrifugation and eluted twice in 500
m
l Buffer E and assayed for
radioactivity. Similarly, if competition binding assays using FIT2 were to be
performed in triplicate using [
3
H]-TAG and unlabeled DAG at a concentration of
100 nM radiolabeled ligand, 30 pmol of radiolabeled [
3
H]-DAG were dried down
with 3-12 nmol of unlabeled DAG, resolubilized, and reacted with FIT2 as described
above. Dose-response curves with and without unlabeled competitor were generated
to demonstrate competition binding at various concentrations of radiolabeled ligand
(
Fig. 11.6
). As a negative control, we demonstrated that FIT2 does not bind choles-
teryl oleate, another neutral lipid. Competition binding assays can be used to explore
the propensity of FIT proteins for neutral lipids with different acyl chain lengths in
varying
sn
positions and of differing degrees and locations of unsaturation.
11.2.2.2
Binding assays between purified FIT2 overexpressed in
HEK293 cells and neutral lipids in detergent micelles
In an effort to determine the protein structural requirements for lipid binding, we con-
structed a saturating scanning-alanine mutagenesis library of FIT2 and developed an
in vitro
high-throughput cell culture-based assay for assessing lipid binding.
HEK293 cells were transfected in triplicate with V5-His
6
tagged murine FIT2
mutants (FIT2-V5) in a pcDNA3.1-V5-His
6
mammalian expression vector. Expres-
sion was allowed to occur for 36-48 h, after which cells were washed off tissue
