Toyopearl hw 40c

To detect the insulin monomer, SEC was performed. The gel for SEC (Toyopearl HW-40C from Tosoh Co., Tokyo, Japan) was filled in the column (bed volume 2 mL) and equilibrated with 20 % acetic acid. The molecular weight exclusion limit of the gel was 2.18 × 10³ Da according to the certificate of analysis. Protein samples (100 μL) were centrifuged as described above, and the resulting supernatants were loaded onto the column. Elution was performed using the acidic solution, and the protein concentration of each fraction (100 μL) was measured using an ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Bovine serum albumin (60 kDa) was used as a calibration marker of molecular weight and eluted in fraction nos. 7–8.

1-Iodobutane, 1-iodooctane, 1-iodododecane, 1-iodohexadecane, and 1-iodooctadecane were purchased from Tokyo Chemical Industry Corporation (Tokyo, Japan). L-Ascorbic acid (AA), L-Ascorbic acid sodium salt (AANa), acetyl chloride, chloromethyl methyl ether, potassium carbonate, hydrochloric acid (HCl), oxatomide, and p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside (PNAG) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Diaion HP20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Wakogel C-200 (FUJIFILM Wako Pure Chemical Corporation), Silica Gel 60 (Merck KGaA, Darmstadt, Germany), and TOYOPEARL HW-40C (Tosoh Corporation, Tokyo, Japan) were utilized for column chromatography. Monoclonal anti-dinitrophenyl antibody (DNP-IgE) and dinitrophenyl-labeled human serum albumin (DNP-HSA) were obtained from Sigma-Aldrich Co., St. Louis, MO, USA. All of the chemicals used were of the highest grade commercially available.