Hrp conjugated goat anti rabbit secondary antibody

Whole-cell protein extracts were prepared using RIPA lysis buffer (PC101, EpiZyme, China). Proteins were separated on a 10% SDS/PAGE gel and electroblotted on to an Immobilon-P membrane (Millipore). The membrane was blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing Tween 20 (TBST) buffer (20 mM Tris/HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), then incubated with the primary antibody overnight at 4°C, washed with TBST buffer, and incubated with HRP-conjugated secondary antibody for 1.5 h at room temperature. After being washed with TBST buffer, blots were developed with SuperSignal West Pico or Femto chemiluminescent substrate (Pierce) or ECL Plus Western blotting detection reagents (GE Healthcare). Antibodies used were rabbit monoclonal to α-tubulin (ab179484, Abcam, Cambridge, U.K.), rabbit monoclonal to EGFP (ab184601, Abcam, Cambridge, U.K.). Proteins recognized by the antibodies were detected by ImageQuant LAS 500 (GE, CT, U.S.A.) using HRP-conjugated Goat Anti-Rabbit secondary antibody (BBI, Sangon Biotech, China).

The EIF4B recombinant protein (10 μg) was separated by a 10% SDS-PAGE and transferred to PVDF membrane. An equal amount of BSA was also loaded as a negative control. The proteins on the membrane were denatured and renatured in AC buffer (100 mM NaCl, 20 mM Tris-HCl pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% non-fat milk and 1 mM DTT) by gradually reducing the guanidine-HCl concentration. After blocking the membrane with 5% non-fat milk in 1 × TBST buffer at RT for 1 h, the membrane was incubated with 1 μg/ml recombinant hnRNPK protein at 4 °C overnight. After that, the membrane was incubated with anti-hnRNPK antibody (Proteintech, China) (1:1000) in the TBST buffer containing 3% milk at RT for 1 h. Finally, the HRP conjugated goat anti-rabbit secondary antibody (Sangon Biotech, China) (1:4000) was incubated for another 1 h and chemiluminescent detection was performed.