Alexa fluor 680 goat anti mouse igg h l

All common chemicals were obtained from Sigma–Aldrich unless otherwise stated. The generation and characterization of the anti-CoA antibody (1F10) was described recently [23 (link)]. For Western blotting, anti-CoA antibody was diluted in Odyssey blocking buffer (0.17 µg/ml) containing 0.01% Tween 20. Secondary antibodies [Alexa Fluor 680 goat antimouse IgG H&L (Life Technologies)] were diluted in Odyssey blocking buffer (1 : 10 000) containing 0.02% sodium dodecyl sulfate (SDS).

The His-Vip3Aa20 protein, MIR162 maize standard substance and negative maize samples, and gradient concentrations of His-Vip3Aa20, His-Vip3Aa1/19, His-Vip3Aa7/10, His-Vip3Aa14 and His-Vip3Aa8 were electrophoresed on a 4–12% nuPAGE gel (Invitrogen, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, US). The membranes were incubated with 5 μg/ml His-Vip3Aa20 mAbs at 4 °C overnight after nonspecific sites were blocked with 5% skim milk. The membranes were washed three times and incubated with Alexa Fluor™ 680 goat anti-mouse IgG (H+L) (Invitrogen, USA) for 1 h at room temperature. The membranes were processed using an Odyssey scanner (LI-COR, USA).

Total brain homogenates (30 μg) in urea were mixed with Laemmli sample buffer and then resolved by SDS–PAGE using 10% or 4–12% Bolt Bis-Tris gels (Invitrogen) at 80 V for 10 min followed by 160 V for 35 min. Proteins were transferred onto nitrocellulose membranes (Invitrogen) using the iBlot 7 min dry transfer system (ThermoFisher Scientific). Membranes were blocked with TBS StartingBlock buffer (ThermoFisher Scientific) for 40 min at room temperature, and then probed overnight at 4°C with primary antibodies diluted in TBS StartingBlock buffer. Membranes were rinsed and incubated with secondary antibodies for 45 min at room temperature. Primary antibodies were Tau-5 (Invitrogen, MA5-12808, 1:1,000 dilution) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam ab8245, 1:2,000), which was used as a loading control. The corresponding secondary antibodies were Alexa Fluor790 donkey anti-mouse IgG (H+L) (Invitrogen) (Invitrogen, A11371, 1:20,000) and Alexa Fluor680 goat anti-mouse IgG (H+L) (Invitrogen, A21058, 1:20,000). Membranes were imaged using an Odyssey Infrared Imaging system (LiCor Biosciences).