title: Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi.
abstract: Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.
authors.affiliation: Max-Planck-Institute for Immunobiology, D-79108 Freiburg, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Hölscher
authors.forename: R
authors.initials: R
authors.lastname: Hartmann
authors.forename: H
authors.initials: H
authors.lastname: Mossmann
authors.forename: G A
authors.initials: GA
authors.lastname: Schaub
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis / Q000737:chemistry; D000954:Antigens, Surface / Q000032:analysis / Q000737:chemistry; D015153:Blotting, Western; D002498:Centrifugation; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D008565:Membrane Proteins; D015800:Protozoan Proteins; D012710:Serum Albumin, Bovine