Cm sepharose ff

The sources of the chemicals were described previously [16 (link)]. Leaves from elder were harvested at Cobos de Cerrato (Palencia, Spain) in early summer. CM-Sepharose FF, Q-Sepharose FF, CM-Sepharose FF, Sepharose 6 B, and Superdex−75 HiLoad 26/60 columns were purchased from GE Healthcare (Barcelona, Spain). The acid-treated Sepharose (AT-Sepharose) was prepared as described in [66 (link)], treating the Sepharose 6 B with 0.1 N HCl at 50 °C for 3 h. The gel was then washed with water (Elix 5, Millipore) until a neutral pH was obtained, and stored in water at 4 °C until it was used. Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin was purchased from Merk Life Science S.r.l. (Milan, Italy). Acetonitrile (CH3CN), formic acid (FA), and water (LC–MS grade) were from Fisher Scientific Italia (Milan, Italy). Century™-Plus RNA Markers were purchased from Fisher Scientific (Madrid, Spain). Rabbit reticulocyte lysate system (nuclease-treated) was purchased from Promega Biotech Iberica S.L. (Alcobendas, Madrid, Spain).

The excised small intestines were rinsed with 0.02 M K₂HPO₄-KH₂PO₄ buffer at pH 7.5 and homogenized with the same solution before centrifugation at 15,000× g at 4 °C for 30 min. The supernatant was centrifuged at 105,000× g at 4 °C for 60 min. Ammonium sulfate was added to the obtained supernatant to yield precipitated protein. The concentration of ammonium sulfate was increased stepwise, and the precipitate was recovered at each stage. The protein precipitated by 60–80% ammonium sulfate was collected and dialyzed against 0.02 M K₂HPO₄-KH₂PO₄ buffer. The dialyzed solution was purified with cation-exchange chromatography (CM Sepharose FF, GE Healthcare, Chicago, IL, USA), and the obtained fraction was mixed with 50 mM ACSO in 0.02 M K₂HPO₄-KH₂PO₄ buffer at 30 °C for 30 min in a sealed vial. Volatiles in the headspace were collected by solid-phase microextraction using divinylbenzene/carboxen/polydimethylsiloxane fiber (57328-U, Sigma-Aldrich, St. Louis, MO, USA) for 30 min while the solution was continuously stirred. The absorbed volatiles were analyzed by GC-Atomic Emission Detector (HP 6890GC, HP G2350A AED, Agilent technology, Santa Clara, CA, USA) equipped with analytical column (DB-1, J&W Scientific, Folsom, CA, USA). Sulfuric compounds were detected at 181 nm, and carbon compounds were detected at 193 nm.