Simple affinity purification of antibodies using in vivo biotinylation of a fusion protein.
We describe a method for affinity purification of antibodies using gene fusions to the antigen. Fusions are made to a protein domain which is recognized in vivo by biotin ligase and biotinylated at a unique position in the fusion protein. When expressed in E. coli, the fusion protein can be captured from a whole cell lysate using an avidin resin to generate an affinity column which is useful for antibody purification. The procedure is simple, rapid and does not require chemical biotinylation or prior purification of the target antigen.
Lesley
S A
SA
Promega Corporation, Madison, WI 53711, USA. slesley@promega.com
Groskreutz
D J
DJ
D000906:Antibodies / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D000915:Antibody Affinity / Q000276:immunology; D019677:Artificial Gene Fusion / Q000379:methods*; D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D010455:Peptides / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins; D014157:Transcription Factors
D000906:Antibodies; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; D012097:Repressor Proteins; D014157:Transcription Factors; D001360:Avidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli