シクロヘプタアミロース-グアニジン系に溶解したフルオレセインイソチオシアネートを用いるたん白質のメンブランフィルター上でのけい光分析

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A sensitive, specific, and simple method for the microassay of protein on a membrane filter is described. In the present study, fluorescein isothiocyanate (FITC) was found to be readily soluble in cycloheptaamylose solution in 8 M guanidine and the resultant mixture was employed in the fluorescent staining of a protein spotted on a membrane filter. Reagent: Five milligrams of FITC is dissolved in 0.5 ml of 8 M guanidine hydrochloride containing 100 mg cycloheptaamylose. Procedure: A sample solution containing 0.5 μg to 4 μg of protein is made 0.1 M with respect to magnesium chloride prior to the assay. When the volume of the sample solution is not more than 2 μl, the sample is spotted with the aid of micropipet on Sartorius Membranfilter SM 113 of 10 mm wide. When the volume is in the range of 2 μl to 2 ml, the protein is collected by filtering the sample solution through the membrane filter employing the suction device illustrated in Fig. 1. The spotted membrane is immersed into the reagent mentioned above and allowed to stand for 30 min at room temperature. The membrane is then rinsed three times with agitation for each 1 min in 0.1 M sodium tetraborate. The fluorescence intensity of the spot is measured with Yamato Scanning Fluorometer, Model SFR-21. Results: The standard curve for bovine serum albumin was linear in the range of 0.5 μg to 4 μg. Amines, amino acids, and other substances of low molecular weight tested did not affect the assay. The reagent herein described may also be applicable to the fluorescent labeling of protein in solution.
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シクロヘプタアミロース-グアニジン系に溶解したフルオレセインイソチオシアネートを用いるたん白質のメンブランフィルター上でのけい光分析

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