Goat anti rat igg hrp

Tissues were homogenized in RIPA buffer (Sigma). Equal amounts of protein were loaded into a 4%-12% NUPAGE gel (Fisher). The proteins in the gel were transferred onto PVDF membranes (Invitrogen) using the Invitrogen iBlot system. The top half of the membranes (containing IgG) were blocked overnight at 4°C in 5% Blocking Grade Blocker (Bio-Rad) and 0.1% Tween-20 (Bio-Rad) in PBS (VWR). The membrane was then incubated with goat anti-Rat IgG-HRP (Bio-Rad, 5204-2504, 1:10,000) for 1 hour at room temperature. The lower half of the membrane (containing GAPDH) was blocked for 2 hours at room temperature and incubated overnight at 4°C with the primary antibody, mouse anti-GAPDH (6C5) (Calbiochem, 1:10 000). After washing, the membrane was incubated 30 minutes with the secondary antibody, goat anti-Mouse-HRP (Cell Signaling, 7076P2, 1:10 000). After the last antibody incubation, both membranes were washed and incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, 34095) and imaged using an iBright FL1000 imager (Invitrogen).
Plasma zonulin, a marker for gut barrier integrity, was measured using a rat zonulin ELISA kit (MyBioSource, MBS753035) according to the manufacturer’s protocol.