title: Purification and electrophoretic characterization of bovine conglutinin.
abstract: In this study a two-stage procedure for purification of conglutinin using affinity and ion-exchange chromatography was developed. To isolate conglutinin from bovine serum, its unique ability to bind to complement component iC3b was exploited. Incubation of bovine serum with chromatographic beads (TSK, Toyopearl HW-75 F) at 37 °C allows for iC3b deposition and subsequent binding of conglutinin. A single protein fraction eluted with ethylenediaminetetraacetic acid (EDTA) was then separated on an ion-exchange column in an NaCl gradient. The purification was evaluated by SDS-PAGE and western blotting. Conglutinin analyzed by SDS-PAGE under reducing conditions showed two main bands at 41 and 47 kDa and eight weaker bands. Nonreduced conglutinin appeared as a ladder pattern composed of many fractions ranging from 34 to 630 kDa. The bands at 34, 153, 174, 247, 338 and 387 kDa displayed the highest optical density. In the native conglutinin profile four fractions were observed, and the pI of this protein was below 8.5. The presence of sugar residues in the conglutinin molecule was detected using Schiff's reagent.
authors.affiliation: University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland. marta.dec@up.lublin.pl
authors.forename: Marta
authors.initials: M
authors.lastname: Dec
authors.forename: Andrzej
authors.initials: A
authors.lastname: Wernicki
authors.forename: Andrzej
authors.initials: A
authors.lastname: Puchalski
authors.forename: Renata
authors.initials: R
authors.lastname: Urban-Chmiel
authors.forename: Marcin
authors.initials: M
authors.lastname: Grąz
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D037222:Collectins / Q000302:isolation & purification* / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D012712:Serum Globulins / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D037222:Collectins; D012712:Serum Globulins; C024014:conglutinin; D012965:Sodium Chloride; D003179:Complement C3b