Bt Research 2012, Vol.3, No.4, 20
-
28
26
toxin was biotinylated using biotinyl-N-hydroxysucc-
inimide ester (GE Helthcare) following the
manufacturer's instructions. Binding of 10 nmol/L
labelled toxin to 10 µg of
A. grandis
BBMV total
protein was carried out in 100 µL binding buffer (1×
PBS , 0.1% BSA, 0.1% Tween 20, and pH 7.6). After
1
h at 25
℃
,
unbound toxin was removed by
centrifugation (10 min at 14,000×
g
).
The pellet
containing the BBMV with bound toxin was washed
twice with 100 µL of the same buffer and finally
suspended in 1X PBS, pH 7.6. An equal volume of
2
X sample loading buffer (0.125 mol/L Tris–HCl, pH
6.8, 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol,
and 0.01% bromophenol blue) was added, samples
were boiled for 3 min, the proteins separated in a
SDS-PAGE gel (10%) and transferred to a
nitrocellulose membrane using the Trans-Blot
®
SD
Semi-Dry Electrophoretic Transfer Cell following the
manufacturer’s instructions (Bio-Rad). The biotinylated
protein was visualized by incubation with streptavidin
conjugated with peroxidase (ECL, GE Helthcare)
(1:4,000
dilution) for 1 h, followed by incubation
with luminol (ECL, GE Helthcare). For the
competition binding assay, 10 nmol/L labelled toxin
plus 1000 nmol/L unlabelled toxin were incubated
with 10 µg of
A. grandis
BBMVs total protein in 100 µL
binding buffer.
3.7
Bioassays
Five doses of recombinant Cry10Aa crystals purified
by centrifugation in sucrose gradients (100 µg/mL,
50
µg/mL, 20 µg/mL, 10 µg/mL and 5 µg/mL), IPS82
strain (1.5 µg/mL, 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL,
0.1
µg/mL) and S1804 strain (1.5 µg/mL, 1.0 µg/mL,
0.5
µg/mL, 0.25 µg/mL, 0.1 µg/mL) were separately
added to
A. grandis
artificial diet as described by
(
Martins et al., 2010). Each protein dose was added to
5
mL of the artificial diet before it was poured out into
six well cell culture plates (TPP, Techno Plastic
Products AG, Switzerland). Five holes were punched
in each well and each hole received a
A. grandis
neonate larva. The bioassay was kept in an incubator
with photoperiod of 14 h/10 h (light/dark) at 27
℃
.
Mortality was recorded seven days later, and the LC
50
obtained by Probit analysis (Finney, 1971).
Acknowledgement
This work was supported by University of Brasília, Graduate
Program in Molecular Biology. It was financed by CAPES
(
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior),
CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico), FINATEC (Fundação de Empreendimentos
Científicos e Tecnológicos) and FAP-DF (Fundação de Apoio a
Pesquisa do Distrito Federal).
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