[31]
IMMUNOAFFINITYPURIFICATION
301
[31] O n e - S t e p I m m u n o a f f i n i t y P u r i f i c a t i o n o f T r a n s f e r r i n
By EDWARD A. BAYER and MEIR WILCHEK We have previously promoted the use of avidin-biotin technology for improving the versatility of affinity chromatography. 1 For this purpose, the affinity column would consist of an avidin- or streptavidin-containing resin to which various biotinylated binders can be bound. The immobilized binder would then be used for the purification of its molecular counterpart. A major advantage of this approach would be that following the preparation of the initial avidin-containing affinity matrix, additional chemical manipulations would not be necessary. In this chapter, we present procedures for the immobilization of a biotinylated antibody specific for transferrin. The column can be used for the one-step isolation of transferrin directly from plasma.
Immobilization of Biotinylated Antibody on Avidin-Containing Column Reagents Avidin-containing affinity resin, 2 - 1 . 5 mg protein/ml Sepharose Biotinylated rabbit anti-(human)-transferfin antibody (polyclonal)3 Phosphate-buffered saline (pH 7.4, PBS) 0.1 M Acetic acid Procedure. A solution (1 ml) containing 1.7 mg biotinylated antitransferrin antibody is applied to a 1-ml avidin column. The column is washed with 5 column volumes of PBS or until the A280reaches a minimum value (
IMMUNOAFFINITYPURIFICATION
301
[31] O n e - S t e p I m m u n o a f f i n i t y P u r i f i c a t i o n o f T r a n s f e r r i n
By EDWARD A. BAYER and MEIR WILCHEK We have previously promoted the use of avidin-biotin technology for improving the versatility of affinity chromatography. 1 For this purpose, the affinity column would consist of an avidin- or streptavidin-containing resin to which various biotinylated binders can be bound. The immobilized binder would then be used for the purification of its molecular counterpart. A major advantage of this approach would be that following the preparation of the initial avidin-containing affinity matrix, additional chemical manipulations would not be necessary. In this chapter, we present procedures for the immobilization of a biotinylated antibody specific for transferrin. The column can be used for the one-step isolation of transferrin directly from plasma.
Immobilization of Biotinylated Antibody on Avidin-Containing Column Reagents Avidin-containing affinity resin, 2 - 1 . 5 mg protein/ml Sepharose Biotinylated rabbit anti-(human)-transferfin antibody (polyclonal)3 Phosphate-buffered saline (pH 7.4, PBS) 0.1 M Acetic acid Procedure. A solution (1 ml) containing 1.7 mg biotinylated antitransferrin antibody is applied to a 1-ml avidin column. The column is washed with 5 column volumes of PBS or until the A280reaches a minimum value (
