Uv 2077 detector

The SEC-MAL system consists of a P900 HPLC pump (GE), a UV-2077 detector (Jasco) and a Tri Star Mini Dawn light scattering instrument (Wyatt). 100 μl of purified WT UraA or UraA variants proteins, each at 1 mg/ml, was injected into a WTC-030S5 gel filtration chromatography and eluted isocratically at 0.5 ml/min in a buffer containing 25 mM Tris pH 8.0, 150 mM NaCl, 1 mM Uracil, 0.2% DM. Data collection and analysis was performed with Astra 6 software (Wyatt).

Light and heavy versions of the selected peptides were synthetized using standard F-moc chemistry. We used ¹³C and ¹⁵ N lysine and arginine for SIL heavy peptides, resulting in an 8- and 10-Da mass shift, respectively, compared with their light counterparts. Cys residues were blocked with 50 mM iodoacetamide for 1 h at 37 °C. Synthesized peptides were purified on a C18 reversed phase column with a 0–65% ACN gradient at 2 ml/min (JASCO Pu-2089 Plus pump coupled to a JASCO UV-2077 detector). The UV detector was set at 214 and 280 nm to monitor eluting peptides. Main chromatographic peaks were collected and re-analyzed for purity assessment by high resolution analytical LC on a Scharlau C18 column (5- μm particle size, 2 mm I.D. × 25 cm) using a 40-min 0–70% ACN gradient at 250 μl/min at 40 °C (Ultimate 3000 HPLC). The minimal purity of all peptide preparations was set as > 90% pure. Molecular weight and peptide purity were further assessed by MALDI TOF/TOF analysis (SCIEX 4800). Peptide quantification was done by amino acid analysis at the Protein Chemistry core facility of the Biological Research Center (CIB-CSIC; Madrid, Spain).