Rapid and efficient isolation of transferrin and ferritin from Manduca sexta

Joy Winzerling, Patricia Nez, Jerker Porath, John H. Law

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)217-224
Number of pages8
JournalInsect Biochemistry and Molecular Biology
Volume25
Issue number2
DOIs
StatePublished - 1995

Fingerprint

Manduca
Hemolymph
Manduca sexta
ferritin
transferrin
Transferrin
Ferritins
hemolymph
ultracentrifugation
Ultracentrifugation
Gels
gels
Purification
iron
Iron
Iron-Binding Proteins
Affinity chromatography
Ion chromatography
Proteins
Collodion

Keywords

  • Ferritin
  • Immobilized metal ion affinity chromatography
  • Iron-binding proteins
  • Transferrin

ASJC Scopus subject areas

  • Insect Science
  • Biochemistry
  • Molecular Biology

Cite this

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta. / Winzerling, Joy; Nez, Patricia; Porath, Jerker; Law, John H.

In: Insect Biochemistry and Molecular Biology, Vol. 25, No. 2, 1995, p. 217-224.

Research output: Contribution to journalArticle

Winzerling, Joy ; Nez, Patricia ; Porath, Jerker ; Law, John H. / Rapid and efficient isolation of transferrin and ferritin from Manduca sexta. In: Insect Biochemistry and Molecular Biology. 1995 ; Vol. 25, No. 2. pp. 217-224.
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