Gellan beads as a transparent media for protein immobilization and affinity capture.
Gellan gum beads are presented as a novel substrate for protein immobilization and immobilized protein activity measurements. The optical transparency of the gellan beads down to 200 nm provides a method for direct quantitation of the amount of protein immobilized onto the beads. The ability to utilize these beads in a non-aqueous activation step allowed for a fourfold increase in the amount of protein immobilized, and this method was used to immobilize Protein A onto gellan beads at a final yield of 1.42+/-0.07 mg of Protein A/g of beads. The optical transparency also allowed for detection of the activity of the immobilized Protein A simply by measuring the absorbance of the beads following capture of rabbit IgG. This activity measurement method was compared with a traditional method utilizing the amount of protein remaining in solution after the IgG capture step. The traditional method yielded an activity measurement of 10.9+/-0.2 mg IgG/mg of Protein A, while the absorbance method showed an activity of only 7.5+/-0.3 mg IgG/mg of Protein A. The difference can be explained by the more direct measurement used in the absorbance method. The optical transparency of the beads was also evaluated in a fluorescence based IgG capture experiment, showing that detection of fluorescent IgG captured on the beads was possible with no interference from the beads.
Ferrance
Jerome P
JP
Department of Chemistry, McCormick Road, University of Virginia, Charlottesville, VA 22904, USA. jpf3p@virginia.edu
D002846:Chromatography, Affinity / Q000379:methods*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008863:Microspheres; D011135:Polysaccharides, Bacterial; D013205:Staphylococcal Protein A / Q000737:chemistry
D007074:Immunoglobulin G; D011135:Polysaccharides, Bacterial; D013205:Staphylococcal Protein A; C048288:gellan gum