Amberlite xad4

Analytical TLC was performed on Merck Kieselgel 60 F254 or RP-8 F254 plates of 0.2 mm layer thickness. The plate was visualized by UV light or by spraying with H₂SO₄/AcOH/H₂O (1:20:4), followed by heating at 120 °C for about 1 min. The plates were then heated for 5 min at 120 °C. Preparative TLC was performed on Merck Kieselgel 60 F254 plates, of 0.5 or 1 mm film thickness. Column chromatography (CC) was performed on Fluka (Seelze, Germany) Amberlite XAD-4, on Merck Kieselgel 60 (70–240 µm) and Merck Kieselgel 60 (40–63 µm), or on Baker (Deventer, The Netherlands) RP-18.

Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1-¹⁴C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl-³H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy. All the other reagents were of analytical grade.

E. coli Rosetta(DE3)pLysS cells were from Novagen (Rome, Italy); His Trap HP and PD10 columns were from GE Healthcare; l-[³H]histidine was from American Radiolabeled Chemicals (ARC Inc., USA); C₁₂E₈, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), cholesterol, Sephadex G-75, imidazole, l-histidine and all the other reagents were from Merck KGaA (Germany).

His Select Nickel affinity gel (P6611), Monoclonal Anti-polyHistidine-Peroxidase antibody (A7058), Amberlite XAD-4 (06444), egg yolk phospholipids, cholesterol (C3045), Thrombin (605,157), n-Dodecyl β-D-maltoside (D4641), Sephadex G-75 (G75120), L-glutamine (G1626) from Merck Life Science; Isopropyl β-D-1-tiogalattopiranoside (IPTG, A4773) from AppliChem; restriction endonucleases, T4 DNA ligase (EL0014), and Phusion™ High-Fidelity DNA Polymerase (F530S) from ThermoFisher scientific; MegaMan Human Transcriptome Library, BL21 codon plus RIL strain from Agilent technologies; Lemo21(DE3) strain from New England Biolabs; C41(DE3) from Lucigen. Pico-Fluor Plus and L-[³H]-glutamine from Perkin Elmer; pET-28a-Mistic (#85,999) from Addgene.

All the solvents, including diethyl ether, methanol, ethyl acetate, and n-hexane were purchased by Levanchimica srl (Bari, Italy). Amberlite® XAD-4, thin-layer chromatography (TLC, silica gel plates Merck 60 F254), and silica gel (0.040−0.063 and 0.063−0.200 mm) were purchased from Merck (Darmstadt, Germany).

Two XK 50 columns, (50 mm diameter; 250 mm length) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used of which the first column was loaded with 350 g of Amberlite XAD4 and the second with 400 g of Amberlite IRA958 (Sigma-Aldrich, Saint Louis, MO, USA) [27 (link)]. The flow rate in these columns was 1 mL/min (GP 50 gradient pump, Thermo Fisher Scientific Inc., Waltham, MA, USA) for both the adsorption and wash sequence. After a solution had been pumped through a column, the column was emptied by purging with air using a hose pump. XAD4 was cleaned with 700 mL of 10% ethanol solution and subsequently washed with 1400 mL of deionized water. 5750 g of the AC solution was passed through the XAD4 column after which a sample of 250 g was removed from the adsorption column permeate. The second column (IRA958) was washed with 1400 mL of deionized water and 5500 g of permeate was passed through this column, producing the purest permeate in this series, which was designated adsorption preparation (AD). The yield for each purification step was calculated based on the galactoglucomannan content (g) before and after each purification step and was expressed as % yield. The purification scheme is illustrated in Figure 6.

E. coli Rosetta(DE3)pLysS cells were from Novagen (Rome, Italy); ECL plus, Hybond ECL membranes were from GE Healthcare; l-[³H]Arg was from Perkin Elmer (Waltham, MA, USA); conjugated anti-His6 antibody, TX-100, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), Sephadex G-75, His-Select resin, l-Arg and all the other reagents were from Sigma-Aldrich (Saint Louis, MO, USA).