Rapid and efficient isolation of transferrin and ferritin from Manduca sexta.
Rapid and efficient isolation of transferrin and ferritin from Manduca sexta.
复制标题
从 Manduca sexta 中快速有效地分离转铁蛋白和铁蛋白。
DOI：
10.1016/0965-1748(94)00058-p
复制
发表时间：
1995
影响因子：
3.8
通讯作者：
Law,JH
中科院分区：
文献类型：
--
作者：
Winzerling,JJ;Nez,P;Porath,J;Law,JH
We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.
