Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8419748 | Journal of Immunological Methods | 2010 | 5 Pages |
Abstract
Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37 °C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 μg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (Kd = 2.3 â 3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.
Keywords
CL-43TBSTRIFMAHBSMBLhepes buffered salineEDTAethylenediamine tetra-acetic acidsodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGETris-buffered salineTime-resolved immunofluorometric assaySPRSurface plasmon resonancecollectin-43CollectinComplement activationMannan-binding lectinC-type lectinPurificationConglutinin
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Authors
Thomas Krogh-Meibom, Klaus Lønne Ingvartsen, Ida Tornoe, Nades Palaniyar, Anthony C. Willis, Uffe Holmskov,