Trans blot turbo midi pvdf

Biotinylated protein levels in mouse tissues were assessed by immunoblotting. Tissue lysates were denatured at 95 °C for 5 minutes in 4X Laemmli sample buffer with 10% β-mercaptoethanol. Samples were loaded on Criterion™ TGX™ any KD gels and run in Novex® Tris-Glycine SDS running buffer. Samples were then transferred to Trans-Blot^® Turbo™ Midi PVDF using the Bio-Rad rapid semi-dry system. After Ponceau S staining, membranes were blocked in 2% non-fat milk in Tris-buffered saline with Tween-20^® for 1 hour at room temperature. Membranes were incubated overnight at 4 °C with the following primary antibodies at 1:5,000 dilution in blocking solution: Rabbit polyclonal anti-biotin (ab1227, Abcam), rabbit polyclonal anti-transferrin (ab82411, Abcam), rabbit polyclonal anti-BSA (A11133, ThermoFisher Scientific), rabbit polyclonal anti-GAPDH (ab9485, Abcam), rabbit polyclonal anti-beta tubulin (ab6046, Abcam). Goat anti-rabbit HRP secondary antibody or Pierce™ high sensitivity streptavidin-HRP antibody (21130, ThermoFisher Scientific) was added at 1:10,000 dilution in blocking solution and membranes were incubated for 1 hour at room temperature. Signal was visualized using SuperSignal™ West Dura Extended Duration Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate on HyBlot CL^® film.

Protein extraction was carried out by acetone precipitation (33 (link)). Protein concentration was determined by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) and total protein was stored at −80°C. Sixty micrograms of total proteins were separated by molecular weight in a poliacrilamyde gel [Criterion™ TGX™ Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA)] and transferred to a Trans-Blot^® Turbo™ Midi PVDF membrane by Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was incubated with primary antibody against NF-κB p105/p50 (Abcam, Cambridge, UK; #ab7971, 1:400) overnight at 4°C and a chicken polyclonal antibody against GAPDH (EMD Millipore, #AB2302, 1:1,000). Then, the membrane was incubated with secondary antibodies for 1 h at room temperature. A goat anti-rabbit (R&D Systems, #HAF008, 1:3,000) and a rabbit anti-chicken (Sigma-Aldrich, #A9046-1ML, 1:9,000) were used, respectively. After that, the membrane was developed using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and the images were obtained using the ImageQuant LAS4000 (GE Healthcare Life Science). ImageJ software (NIH, USA) was used for the quantification.