. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 486 Y. TAKEI ET AL. C|AS LSTCVLGKLSQELHKLQTYPRT DV N LSTCVLGKLSQELHKLQTYPRT DV •N LSTCVLGKLSQELHKLQTYPRT NT A|GTP ;TF -NH2 -NH2 -NHj CGNLSTCMLGTYTQD CGNLSTCMLGTYTQD : F NKFHTFPQT NKFHTFPQT S •-. IGVGAP IGVGAP -NH2 -NH2 Fowl CT Eel CT Salmon CT Rat CT Human CT Porcine CT Bovine CT Ovine CT Figure 1. Amino acid sequences of the mammalian and non-mam- malian calcitonins (CTs) that have been sequenced to date. The identical amino acids within the same group are boxed. with 67% and acetone, respectively, at 4°C. The extract
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 486 Y. TAKEI ET AL. C|AS LSTCVLGKLSQELHKLQTYPRT DV N LSTCVLGKLSQELHKLQTYPRT DV •N LSTCVLGKLSQELHKLQTYPRT NT A|GTP ;TF -NH2 -NH2 -NHj CGNLSTCMLGTYTQD CGNLSTCMLGTYTQD : F NKFHTFPQT NKFHTFPQT S •-. IGVGAP IGVGAP -NH2 -NH2 Fowl CT Eel CT Salmon CT Rat CT Human CT Porcine CT Bovine CT Ovine CT Figure 1. Amino acid sequences of the mammalian and non-mam- malian calcitonins (CTs) that have been sequenced to date. The identical amino acids within the same group are boxed. with 67% and acetone, respectively, at 4°C. The extract was then subjected to reverse-phase high perfor- mance liquid chromatography (HPLC) on an ODS-120T column ( X 250 mm; Tosoh, Tokyo) with a linear gra- dient elution from 20% to 80% CH,CN in trifluo- roacetic acid (pH ), and each fraction was examined for the presence of immunoreactive calcitonin by im- munoblotting. Each immunoreactive fraction was finally purified on the same column with a solvent of a different pH (ammonium acetate buifer, pH ). The fractions were lyophilized and a small portion of each, or synthetic salmon calcitonin (1 ng-1 ^g), was dis- solved in 10 ^1 of a mixture of M Na2CO3 (pH ) and methanol (4:1, v/v), and blotted onto an Immobilin PVDF transfer membrane (Millipore Co. Ltd., Tokyo). The membrane was soaked in 100%i methanol for 3 s, and washed 3 times in 10 mM phosphate-buffered saline (pH ) containing Tween 20 (PBST) for 5 min. The membrane was washed twice more in PBST con- taining 1% normal goat serum, and then three times again in PBST. The membrane was then incubated with an antiserum raised against salmon calcitonin (Sasayama et 1989) (1/40,000 dilution) for 2 h at room temperature. The unbound antiserum was removed by three washes in PBST, and the membrane was immunostained with a Vectastain ABC kit (Vector Laboratories, California) ac- cording to the protocol included with the kit. A portion of purified
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
