The plate was then stained with orcinol–sulphuric acid–methanol solution by a colour reaction after heating at 125 °C. After fixation with 0.02% polyisobutylmethacrylate (PIBM) in hexane solvent (90% hexane with 10% chloroform, v/v) for 1 min, the HPTLC was blocked with 0.5% bovine serum albumin (BSA) in PBS (137 mM NaCl, 10 mM Phosphate buffer, 2.7 mM KCl, and a pH of 7.4) with 0.02% tween-20 for 30 min at room temperature. About 22 nM of LEC-8 in 10 ml of PBS buffer (0.5% BSA, pH 7.4) was overlaid with the plate for two hours at room temperature (about 20 °C).
The plate was washed with PBST for four times and incubated with antibodies against LEC-8 in a ratio of 1:1000 overnight. After washing with PBST for four times, the plate was incubated with antibodies anti-Rabbit IgG conjugated with Trichostatin A ic50 alkaline phosphatase in a ratio of 1:1000 for two hours. After washing with PBST for four times, the HPTLC overlay was detected by a colour reaction in an alkaline buffer with NBT and BCIP as substrates. To further assess whether pre-incubation with LEC-8 can reduce or inhibit the binding of Cry1Ac to glycolipid, after HPTLC resolution the glycolipid was first incubated with 22 nM of LEC-8 for 2 h at 20 °C before incubation with 20 nM Cry1Ac for 2 h. The plate was then incubated with antibodies against Cry1Ac
for overnight and the second antibodies for 2 h and developed by using the same method as above. To test whether binding of LEC-8 to insect glycolipids inhibit the glycolipids binding to Cry1Ac, an in vitro competition experiment was performed based Topoisomerase inhibitor on the method of Refs. [4] and [11] with some modifications. In brief, the Nunc Polysorp 96-well flat microplate was equilibrated with 25 μl of methanol. The plate was dried at room temperature followed by application
of 7.5 μl gut glycolipids. The plate was then left at room temperature for completely drying followed by blocking the wells with 100 μl of 0.5% BSA in PBS buffer for 30′ at RT. Three treatments with three replicates Rucaparib solubility dmso were performed: LEC-8 only (concentration from 0 to 140 nM), active Cry1Ac only (concentration from 0 to 140 nM) and the mixture of 20 nM Cry1Ac and variable LEC-8 (from 0 to 900 nM). The three treatments at various concentrations in 100 μl PBS were incubated with glycolipids for two hours at room temperature. After washing four times with 200 μl of PBS, the plate was incubated with 100 μl of the first antibodies (anti-Cry1Ac for treatments Cry1Ac and mixture of LEC-8 with Cry1Ac, anti-LEC-8 for treatments LEC-8) at 1:1000 for one hour at room temperature. After washing four times with 200 μl of PBS, the plate was incubated with 100 μl of the second antibodies (anti-IgG conjugated with alkaline phosphatase) at 1:1000 for one hour at room temperature.