Selective adsorption: a new method for purification of protein A-gold complexes.
Protein gold complexes are prepared by adding gold colloids to cytochemically active proteins in solution. The gold particles of the colloid form complexes with the protein spontaneously, but some of the protein remains uncomplexed. Currently, when protein A-gold complexes are prepared, the uncomplexed protein. A is separated from the complex by ultracentrifugation, which is a lengthy procedure and requires special equipment. This report describes a simple and rapid method for removing uncomplexed protein A from freshly-prepared "crude" protein A-gold at the laboratory bench. In this method, larger gold particles of 15-nm diameter are added to a crude protein A-gold preparation made with smaller particles (e.g.,6nm diameter). The 15-nm particles adsorb uncomplexed protein A preferentially, but do not form complexes with already-formed 6-nm protein A-gold. The adsorbed protein A, attached to the 15-nm particles, can then be sedimented in a bench centrifuge, leaving the purified 6-nm protein A-gold complexes in the supernatant. The stability, immunocytochemical activity, and degree of aggregation of the protein A-gold complexes prepared by this method are comparable to protein A-gold complexes prepared by ultracentrifugation. The method is simple to perform, avoids lengthy purification procedures, and yields complexes with reproducible labelling characteristics.
Lucocq
J
J
Department of Anatomy and Physiology, University of Dundee, UK.
D002498:Centrifugation / Q000379:methods* / Q000592:standards; D003102:Colloids; D006046:Gold / Q000302:isolation & purification* / Q000378:metabolism; D007150:Immunohistochemistry; D010316:Particle Size; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods / Q000592:standards
D003102:Colloids; D013205:Staphylococcal Protein A; D006046:Gold