Evaluation of the Sensitization Rates and Identification of IgE-Binding Components in Wild and Genetically Modified Potatoes in Patients with Allergic Disorders - Genetic Engineering: Recent Developments in Applications - page 101

1% SDS, 0.0025% bromophenol blue, 2.5% β -mercaptoethanol, pH 6.8)
and heated in boiling water for 5 min. Standard markers (4-250 kDa; Novex,
San Diego, CA, USA) and the extracts were loaded on a 4-20% Tris-glycine
gel (Novex). Electrophoresis was done with a Novex X cell™ Mini cell for 90
min at 125 V. The gel was fixed and stained with Coomassie brilliant blue.
For immunoblotting, proteins were transferred onto a polyvinylidene difluo-
ride membrane (PVDF; Millipore Co., Bedford, MA, USA) in transfer buffer
(Tris-base 25 mmol/l, glycine 193 mmol/l, and methanol 20%) using a Bio-
Rad transfer apparatus set at 200 mA for 90 min. The blotted PVDF mem-
brane was sliced into 4-mm widths and then incubated in 5% skim milk in
Tris-buffered saline (TBS)-Tween (TBST) for 1 h to block nonspecific bind-
ing to the membranes. Each membrane slice was incubated overnight at 4°C
with patient or control sera diluted 1:2 v/v with 3% skim milk in TBST, and
then washed with 0.1% TBST for 30 min. Subsequently, the membranes were
incubated with goat anti-human IgE conjugated with alkaline phosphatase
(Sigma) for 1 h at room temperature, washed with TBST, and then developed
with BCIP/NBT alkaline phosphatase substrate (Sigma).
effect of simulated Gastric and intestinal Fluid on the
ige-binding components
Crude extracts were prepared from GM and wild-type potatoes and then
heated at 100°C for 5 min. The intrinsic digestibility of the extracts and the
digestibility of extracts preheated and incubated in SGF or SIF were exam-
ined as previously described [14]. Briefly, SGF digestibility was analyzed by
dissolving 680 µ g of naïve crude extract or preheated extract in 200 µ l of pre-
warmed 100 mmol HCl/l (pH 1.2) and 30 mmol NaCl/l containing 0.32%
(w/v) pepsin A (Sigma). Digestion was conducted at 37°C with continuous
shaking, and aliquots of the digested solution (20 µ l) were withdrawn at 0,
0.5, and 60 min. These aliquots were quickly mixed with 26 µ l of sample
buffer (containing 2.5% 2-mercaptoethanol and 1% SDS) and 6.0 µ l of Na2-
CO3 (200 mmol/l). The mixture was then boiled for 5 min and stored at
-20°C until further analysis. To evaluate the effects of SIF, 680 µ g of the naïve
crude extract or the preheated extract were dissolved in 260 µ l of prewarmed
intestinal control solution (0.05 M KH2SO4, pH 6.8) containing 1.0% (w/v)
pancreatin (pancreatin USP; Sigma). This solution was incubated at 37°C
with continuous shaking. Aliquots (26 µ l) were withdrawn at 1, 90, and 240
min, mixed with 26 µ l of sample buffer (containing 2.5% 2-mercaptoethanol
and 1% SDS), and then boiled for 5 min. SDS-PAGE (12%) and IgE-immu-
noblot analysis were then carried out as described above.