Em 1 econo uv absorbance instrument

Allyl dextran-based size-exclusion gel (Sephacryl S-300, GE Healthcare) was used as stationary phase. The gel column was prepared by filling a 15 cm long column with an appropriate amount of allyl dextran-based size-exclusion gel dilute 1:1 with the following solution buffer: 10 mM KCl, 20 mM TEA-HCl (pH 7.5), and 20 mM MgCl₂. The flow rate of the running buffer was 1 ml/min and the presence of molecules along the flow was monitored by reading the absorbance at 254 nm with the EM-1 Econo UV absorbance instrument (Bio-Rad). The speed of the recording pare was set to 1 cm/min.

The association of recombinant and/or endogenous proteins to ribosomal subunits was investigated by fractionating different samples on sucrose density gradient and then probing each fraction for the presence of the proteins by western blot with specific antibodies. Specifically, at the end of each reaction, the samples were layered on linear 10–30% sucrose gradients containing 10 mM KCl, 20 mM TEA-HCl (pH 7.5), and 20 mM MgCl₂; these were centrifuged in a Beckman SW41 rotor at 4°C and 36,000 rpm for 4 h or at 18,000 rpm for 17–18 h. After centrifugation, the gradients were unloaded while monitoring absorbance at 254 nm with the EM-1 Econo UV absorbance instrument (Bio-Rad). The individual fractions (0.5 ml) were collected in single tubes and precipitated adding 1/100 volume of 2% Na-deoxycholate and 1/10 of trichloroacetic acid 100%, vortexed, and let sit over-night at 4°C. Then, the samples were centrifuged 15′ at 13,000×g, the protein pellets were resuspended in 20–40 μl of 1X Laemmli Sample Buffer, separated by 15% SDS–PAGE, and electroblotted to nitrocellulose membrane. On the basis of the protein analyzed, we probed the membrane with house made rabbit polyclonal antibodies (antibody against aSBDS and aIF6) or a 6x-His Tag monoclonal antibody (Thermo Fisher Scientific).