04% SDS, 20% methanol and Tris-HCl, pH 8.0) at a constant voltage of 30 V for the first hour at 4 °C and then at 80 V for 2 h. The membrane was blocked with 5% skimmed milk in phosphate-buffered saline (PBS), pH 7.4, at 4 °C overnight. The immobilized proteins were probed with rabbit anti-BinB

antibody (1 : 20 000), which was prepared by injecting the fast protein liquid chromatography-purified BinB into a rabbit, for 1 h and goat anti-rabbit IgG conjugated with alkaline phosphatase (1 : 5000) for 1 h. The immunoreactive bands were visualized using an ECL chemiluminescent plus kit (GE Healthcare). Protein inclusions (2–3 mg mL−1) were solubilized by incubation in 25 mM NaOH, 5 mM dithiothreitol at 37 °C for 15 min. Solubilized protein was stepwise dialyzed in 250 vol. of carbonate buffer (50 mM Na2CO3, pH 10.0) with a gradual decrease of NaOH concentrations to 19, check details 13.3, 6.7 this website and 3.4 mM. Each dialysis was performed at 4 °C for 1 h. Finally, the samples were dialyzed three times against 250 vol. of the carbonate buffer. The protein was further purified by gel filtration using a superdex

200HR 10/300 column (Amersham Pharmacia Biotech) and purified protein was kept at −20 °C. In vivo mosquito-larvicidal assays were used to determine the toxicity of mutant toxins against 2nd-instar Culex quinquefasciatus larvae, which were supplied by the mosquito-rearing facility in the Institute of Molecular Biosciences, Mahidol University, Thailand. Equimolar mixtures

of BinA wild-type inclusions and BinB mutant inclusions were diluted in 1 mL of water at several twofold serial dilutions, from 64 μg mL−1 to 0.1250 μg mL−1. Tau-protein kinase These 1-mL dilutions were added to wells in a 24-well tissue culture plate, each well containing 10 larvae. The BinA–BinB wild-type inclusion mixture was used as a positive control, while BinB wild-type inclusions were used as a negative control. After a 48-h incubation period, the mortality of the larvae was recorded. The assays were carried out in duplicate, and at least three independent experiments were performed. The LC50 was then analyzed by Probit analysis (Finney, 1971). A nitrocellulose membrane was cut into strips and was equilibrated in PBS. Various amounts of purified truncated BinA (2.5–20 μg) (Limpanawat et al., 2009) were immobilized on every strip at 25 °C using a Bio-Dot Microfiltration Apparatus (Bio-Rad). The dotted membranes were blocked in 5% skimmed milk at 4 °C overnight. Twenty micrograms per milliliter of purified wild type, or mutant, BinB in 5% skimmed milk was overlaid for 1 h on each strip and subsequently washed with 0.1% Tween-20 in PBS (PBS-T20) three times, for 5 min each time. Bound BinB was detected by probing with rabbit anti-BinB (1 : 20 000) for 1 h.