Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution.
The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.
Lee
Jae-Rin
JR
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.
Hahn
Hwa-Sun
HS
Yu
Jae-Ran
JR
Kim
Seong-Tae
ST
Yang
Jun-Mo
JM
Hahn
Myong-Joon
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D003402:Creatine Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D010957:Plasmids; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D003402:Creatine Kinase