Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.
A method is described for the use of magnetic beads as a solid phase for the immunoprecipitation of labeled proteins. The anti-human transferrin receptor monoclonal antibody OKT-9 has been coupled to sheep anti-mouse IgG1-coated magnetic beads using the crosslinking agent dimethyl pimelimidate. The transferrin receptor is readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography following immunoprecipitation from 35S-labeled cell lysates. When compared with precipitations using OKT-9 coupled to protein G Sepharose the magnetic beads result in fewer nonspecific bands. The protocol described is generally applicable to the identification of labeled proteins. In addition, because magnetic beads are amenable to covalent crosslinking procedures they can be used for the purification of proteins from complex mixtures. Covalently crosslinked OKT-9 sheep anti-mouse IgG1-coated magnetic beads have been used to affinity purify unlabeled transferrin receptor from cell lysates giving comparable purity and yield to transferrin Sepharose isolated transferrin receptor. The major advantages offered by magnetic beads compared to conventional affinity matrices are low nonspecific binding and the rapidity with which the purification can be performed.
Karlsson
G B
GB
Department of Biochemistry, University of Oxford, United Kingdom.
Platt
F M
FM
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008280:Magnetics; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011233:Precipitin Tests / Q000379:methods*; D011990:Receptors, Transferrin / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry
D000911:Antibodies, Monoclonal; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011990:Receptors, Transferrin; D012685:Sepharose