Fluorescent labeling of proteins and a plasma membrane using cycloheptaamylose-dansyl chloride complex

✍ Scribed by Toshio Kinoshita; Fumio Iinuma; Akio Tsuji

• Publisher: Elsevier Science
• Year: 1974
• Tongue: English
• Weight: 317 KB
• Volume: 61
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(74)90431-x

✦ Synopsis

Although Rinderknecht (9) proposed the use of DANS-Cl adsorbed on Celite to avoid the employment of organic solvent, the reaction was still heterogeneous and the degree of labeling (D. L.) was often poor '(5).

This paper deals with the preparation of the complex of DANS-Cl with cycloheptaamylose (C7A) (10) and the application of the product, C7A-DAN%Cl complex (CDC), to the dansylation of proteins and a plasma membrane. Satisfactory labeling was observed without using any organic solvent. Materials. DANS-Cl was the gift of Dr. Y. Fujita, Seikagaku Kogyo Co. C7A was purchased from Hayashibara Biochemical Laboratories, Inc., and once recrystallized from water before use. Bovine serum albumin (BSA, fraction V) , ovalbumin (six times recrystallized) and bovine chymotrypsin were purchased from Wako Pure Chemical Co., Nutritional Biochemicals Corporation and Miles Laboratories, Ltd., respectively. Preparation of CDC. To 300 ml of 2 w/v % aqueous C7A solution was added a solution containing 1.2 g of DANS-Cl in 5 ml of acetone dropwise under stirring at room temperature in the course of 15 min. Yellow precipitate deposited on addition of the chloride solution. The mixture was then left standing in an ice bath for 30 min. The precipitate was collected by centrifugation and dried over PzO, under reduced pressure giving 5.6 g of CDC as a yellow powder. IR in KBr disc showed absorption maxima at 3340(0-H), 2890 (C-H), and 1610 (C=C) 632