Hrp conjugated goat anti rat secondary antibody

Transfected NIH 3T3 cells were washed with PBS and lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40 (Igepal CA-630), 0.5% Na-deoxycholate, 0.1% SDS) supplemented with 1 × protease inhibitor cocktail set (Millipore). Proteins were separated on 10% polyacrylamide gel and transferred onto a PVFD membrane (Millipore). Anti-HA primary antibody (High affinity, #11867423001, Roche, dilution 1:1000) and HRP-conjugated goat anti-Rat secondary antibody (#31470, ThermoFisher Scientific, dilution 1:50,000) were used for signal detection with SuperSignal West Femto Chemiluminescent Substrate (Pierce).

After SDS-PAGE, proteins were transferred to a PVDF membrane (Thermo Fisher Scientific, USA) in a transfer buffer (20% methanol in 1× SDS buffer) at 150 mA for 90 min. The membrane was incubated in blocking solution (5% skimmed milk in TBST buffer 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% Tween20) at 4°C with gentle agitation overnight. The membrane was then incubated for 2 h at room temperature with rat anti-GFP primary antibody (Chromotek, Germany) diluted 1:3000 in blocking solution. Unbound antibodies were washed off by three TBST washes for 10 min each. The membrane was incubated with HRP-conjugated goat anti-rat secondary antibody (Thermo Fisher Scientific, USA) diluted 1:10 000 in blocking solution for 2 h at room temperature. Unbound antibodies were washed off by three TBST washes for 10 min each. Labeling was visualized with Amersham ECL Western Blotting Detection Reagent (Cytiva, Germany) using G:BOX.

Frozen colon tissue samples were lysed in RIPA Buffer and the total protein concentration was quantified using a BCA assay kit as per manufacturer’s protocol (Thermo Scientific). Lysates were obtained from three independent biological samples per group and pooled into a single sample for analysis. Polyacrylamide gel electrophoresis was performed to separate proteins using 4–20% stain-free TGX gels (Bio-Rad). Proteins were visualised by UV-induced fluorescence using a Chemidoc imaging system (Bio-Rad) to verify equal loading of samples. The most abundant protein band in each loading control sample was used for quantification. Proteins were transferred to nitrocellulose membranes in a trans-blot turbo transfer system (Bio-Rad). Membranes were blocked for 30 min using 5% skimmed milk in PBST and then probed with rat anti-GFAP primary antibody (1:2000, cat # 13-0300, ThermoFisher) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-rat secondary antibody (1:5000, cat # 31460, ThermoFisher) for 2 h at room temperature and visualisation using enhanced chemiluminescence (ECL kit, GE Healthcare Life Sciences). Data were analysed using the gel analysis package in FIJI^{66 (link)}.