5.5. Protein extraction from insect and plant tissues and western blots

For western blots with insect protein extracts, whole bodies of Hessian fly at different developmental stages were collected and frozen in liquid nitrogen immediately. The frozen insects were ground to powder with a high‐speed electric motor. Equal amounts of ground samples (200 mg) were solubilized into a cold TCA‐2ME‐acetone solution. Protein precipitates were collected by centrifugation. After washing with cold TCA‐2ME‐acetone solution, the protein precipitates were air‐dried and then dissolved into R2D2 buffer containing 7 M urea, 2 M thiourea, 2% 3‐[(3‐cholamidopropyl) dimethyl‐ammonio]‐1‐propane‐sulfonate, 2% N‐decyl‐N,N‐dimethyl‐3‐ammonio‐1‐propane‐sulfonate, 20 mM dithiothreitol, 8 mM Tris(2‐carboxyethyl) phosphine). The samples were stored at −20°C until later use for western blot analyses.

For wheat protein extracts, 10 mm wheat sheaths at the feeding/attack site were collected, and leaf‐sheaths were soaked in 10 ml TE‐SDS buffer (50 mM Tris and 2 mM EDTA, pH 8.0, with 0.1% SDS). Hessian fly larvae that fell into the buffer were removed and the solution containing proteins without any insects was transferred to a new tube. Control samples without infestation were collected in the same way. The solution with proteins was frozen in liquid nitrogen immediately. After several collections following the same way, solutions containing proteins were combined, dialyzed against DI water, lyophilized, and dissolved into sample buffer.

Proteins dissolved in sample buffer were boiled for denaturation. Equal amounts of samples were loaded onto a 12% precasted SDS‐PAGE (Life Technologies). The samples were separated by running the gel at 80 volts for 60 minutes. After separation, proteins on the gel were transferred onto a nitrocellulose membrane using an electric device from Thermo Fisher. After transfer, the membrane was blocked with 5% milk at room temperature for 1 hr, and incubated with an antibody at 1:10,000 dilution in PBST buffer for two hours. The membrane was then washed with 1% PBST for 3 hr with a buffer change every 30 minutes. The membrane was then incubated for 1 hr with an HRP‐conjugated secondary antibody at 1:1,000 dilution (Amersham, GE Healthcare Life Sciences). The membrane was washed again with PBST buffer for 3 hr with a buffer change every 30 minutes. Chemiluminescence was developed using a WesternSure^(R) PREMIUM Chemiluminescent Substrate. Images were visualized with a C‐DiGit Blot Scanner (Li‐Cor).