Amicon ultra spin concentrator

Ribosomal subunits were purified from HeLa cell pellets (National Cell Culture Center). HeLa cells were lysed in Triton-X lysis buffer (15-mM Tris-HCl (pH 7.5), 300-mM NaCl, 1% (v/v) Triton X-100, 6-mM MgCl₂, 1-mg/ml heparin) and the supernatant was subjected to brief centrifugation to remove cellular debris. The supernatant was applied to a 30% (w/w) sucrose cushion containing 0.5-M KCl and centrifuged at 100,000 g to pellet ribosomes. Ribosomes were resuspended in buffer B (20-mM Tris-HCl (pH 7.5), 6-mM magnesium acetate, 150-M KCl, 6.8% (w/w) sucrose, 2-mM DTT) and subsequently treated with puromycin to dissociate ribosomes from the mRNAs. KCl was added to obtain a final concentration of 0.5 M. The dissociated ribosomes were resolved on a 10–30% (w/w) sucrose gradient where the peaks corresponding to the free 40S and 60S subunits were detected by measuring the absorbance at 260 nm. The corresponding fractions were collected, pooled and concentrated in buffer C (20-mM Tris-HCl (pH 7.5), 0.2-mM ethylenediaminetetraacetic acid (EDTA), 10-mM KCl, 1-mM MgCl2, 6.8% (w/w) sucrose) using Amicon Ultra spin concentrators (Millipore). The resultant concentrations of the ribosomal subunits were determined by spectrophotometry using the conversions 1 A260 nm = 50 nM and 1 A260 nm = 25 nM for the 40S and 60S subunits, respectively.

DNA templates for in vitro transcription were amplified by PCR using custom DNA primers (IDT) and Phusion Hot Start polymerase (New England BioLabs). Transcription reaction mixtures (2.5 ml) were assembled using 1,000-µl PCR reaction mixtures as the template (∼0.2 µM template DNA, a 6 mM concentration of each NTP, 60 mM MgCl₂, 30 mM Tris [pH 8.0], 10 mM dithiothreitol [DTT], 0.1% spermidine, 0.1% Triton X-100, T7 RNA polymerase, and 2 µl RNasin RNase inhibitor [Promega]) and incubated overnight at 37°C. After inorganic pyrophosphates were precipitated by centrifugation, the reaction mixtures were ethanol precipitated and purified on a 7 M urea–8% denaturing polyacrylamide gel. RNAs of the correct size were excised, eluted overnight at 4°C into ∼40 ml of diethylpyrocarbonate (DEPC)-treated Milli-Q filtered water (Millipore), and concentrated using Amicon Ultra spin concentrators (Millipore). Mutations were introduced using mutagenized custom DNA reverse primers (Table 3).

FGF21 proteins were buffer exchanged into 20 mM Tris, 8.5% sucrose at pH 7.5, and were concentrated using Amicon Ultra spin concentrators (Millipore). Protein concentrations were determined using a SOLO-VPE Spectrophotometer (C Technologies Inc). The buffered protein samples were incubated at 25 °C and 4 °C up to four weeks, with time points taken at day 0, 2, 7, 14, 28 days. At each time point, the incubated samples were analyzed on Agilent 1100 HPLC system with YMC-Pack Diol 200 (YMC) analytical size exclusion chromatography column and PBS supplemented to 400 mM NaCl as running buffer. Viscosity was determined using a MCR-302 rheometer (Anton Paar) with a CP25 measuring system. The data was analyzed using the Rheoplus V 3.62 software.