Biology Reference
In-Depth Information
No competitor
10 nmol DAG
60
50
40
30
20
10
0
0 0 0 0 0 0 0
pmol [
3
H]-DAG added
FIGURE 11.6
Lipid-binding specificity is illustrated by dose-response competition binding assays. Varying
concentrations of [
3
H]-radiolabeled ligand ([
3
H]-DAG shown here) with excess unlabeled
ligand were reacted with FIT to produce competition dose-response curves. Data are
represented as mean
SD. This experiment was replicated twice with similar results.
Adapted from
Gross et al. (2011)
.
6 h at 4
C in 10% Fos-choline 13 in
Buffer B (10-fold higher concentration of Fos-choline 13 compared to extraction per-
formed from insect cells). Lysates were then clarified at 16,000
culture plates twice in PBS and extracted for
g
at 4
C for 10 min
and supernatants were added to 30
m
l Protein A/G PLUS-Agarose beads (Santa Cruz
Biotechnology) and 3
m
g anti-V5 antibody (AbD Serotec) and overexpressed FIT
proteins and FIT2 mutants from a scanning-alanine mutagenesis library of FIT2 were
immunoprecipitated overnight. The subsequent morning, immunoprecipitated pro-
tein on Agarose beads was washed three times in 800
m
l Buffer C and 100
m
l volume
binding assays were set up and performed as described in
Section 2.2.1.3
. After bind-
ing assays and washes were complete, agarose beads were eluted in 100
m
l SDS
Laemmli loading buffer and 5-10
m
l were subjected to immunoblot analysis with
the remaining 90-95
m
l assayed for
3
H radioactivity. The HEK293 overexpression
and purification system for FIT2 proteins and mutants displayed saturation binding
kinetics and similar TAG and DAG-binding affinity (as determined by half-maximal
binding concentration) and capacity compared to lipid-binding assays performed
using recombinant protein purified from insect cells (
Fig. 11.7
).
11.2.3
Generation of proteoliposomes
In this section, we describe how integral membranes purified in Fos-choline 13
detergent micelles can be used to generate proteoliposomes for further structure-
function studies and the development of reconstitution assays (i.e., attempting to
reconstitute LD formation).
