Immunoaffinity chromatography.
This unit describes the isolation of soluble or membrane-bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. This technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Specifically, antibodies are coupled to Sepharose (an insoluble, large-pore-size chromatographic matrix). High-molecular-weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. To elute the bound antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high- or low-pH buffer. Batch purification of antigens is provided as an alternate procedure that shortens the column loading time. The detergent octyl beta-D-glucoside can be used instead of Triton X-100 for elution. Because octyl beta-D-glucoside has a high critical micelle concentration (CMC), a protocol is provided for its removal by dialysis. The procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method is given in a support protocol.
Springer
T A
TA
Center for Blood Research Harvard Medical School, Boston, Massachusetts, USA.
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003488:Cyanogen Bromide / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D000954:Antigens, Surface; D008565:Membrane Proteins; D012685:Sepharose; D003488:Cyanogen Bromide