Reducing sds sample buffer

Ten microliter translation reactions were performed with 2.8 nM mEGFP mRNA (folded) and 1 μM WT NT-hFMRP as described previously with nLuc mRNA. Forty microliters of 2× reducing SDS sample buffer (Bio-Rad # 1610737) was then added and heated at 70 °C for 15 min. Ten microliters was then separated by standard Tris-Glycine SDS-PAGE (Thermo # XP04200BOX) and transferred on to 0.2 μm polyvinylidene difluoride membrane (Thermo # 88520). Membranes were then blocked with 5% (w/v) nonfat dry milk in TBST (1× Tris-buffered saline with 0.1% (v/v) Tween 20) for 30 min at RT before overnight incubation with primary antibodies in TBST at 4 °C. After three 10 min washes with TBST, membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibody in TBST for 1 h at RT and then washed again with three 10 min washes with TBST. Chemiluminescence was performed with SuperSignal West Pico PLUS for GFP (Thermo # 34577) and with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo # 34095) for tubulin. Blots were imaged using an Azure Sapphire Biomolecular Imager. Rabbit anti-GFP (Cell Signaling # 2956S) was used at 1:1000. Mouse antitubulin (Sigma # T9026-2ML) was used at 1:1000. HRP-conjugated goat anti-rabbit IgG (H + L) (Thermo # 31460) was used at 1:60,000 for GFP and HRP-conjugated goat antimouse IgG (H + L) (Thermo # 31430) was used at 1:10,000 for tubulin.