Immunoaffinity chromatography using electroelution.
Immunoaffinity chromatography and electroelution of protein from solid-phase matrices are two powerful tools often used to purify proteins. In this study, we combined these two techniques and found that antigen was effectively recovered from immunoaffinity resins by electroelution. Yields ranged from 90.5% to 62.8%, with a mean of 74.0 +/- 7.4% (mean +/- standard deviation). A major portion of the eluate, 79.4 +/- 13.1%, was concentrated in a volume of 100 microliters and 94.0 +/- 2.0% was recovered in 200 microliters, even when 1 ml of resin was used. Electroelution had no major effect on the electrophoretic mobility of the antigen. Two distinct antigens, a relatively hydrophilic 230-kDa protein and a hydrophobic 28-kDa protein were successfully electroeluted. In addition two types of immunoaffinity resins, monoclonal antibody covalently linked to CNBr-activated Sepharose or immobilized protein A, were found to be compatible with this method.
Williamson
K C
KC
Laboratory of Malaria Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Duffy
P E
PE
Kaslow
D C
DC
D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000302:isolation & purification*; D001345:Autoradiography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D004563:Electrochemistry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D007457:Iodine Radioisotopes; D015800:Protozoan Proteins