Affinity of the monoclonal antibody M1 directed against the FLAG peptide.
The FLAG (Sigma, St. Louis, MO, USA) peptide is a frequently used hydrophilic and immunogenic fusion tag which was specifically designed to facilitate rapid purification by immunoaffinity chromatography. The monoclonal antibody M1 recognizes the free N-terminus of the peptide tag in a calcium dependent manner. Dissociation of the complex can be performed by the addition of chelating agents such as EDTA. This effect can be exploited for immunoaffinity purification of FLAG-tagged fusion proteins. Kinetic information obtained from monitoring interactions in real-time measurement (Biacore 2000) using surface plasmon resonance as detection principle did not show any difference for association and dissociation rate constants in the presence (k(a) = 3.03 x 10(3) M(-1) k(d) = 1.25 x 10(-3) s(-1)) and in the absence of Ca2+ (k(a) = 3.59 x 10(3) M(-1) s(-1), k(d) = 1.16 x 10(-3) s(-1)). These findings corroborate the reports from Mol. Immunol. 33 (1996) 601-608 describing similar binding analyzed by enzyme-linked immunosorbent assay experiments. These investigations are in contrast to the observations in immunoaffinity chromatography with immobilized anti-FLAG antibody M1.
Einhauer
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Institute of Applied Microbiology, University of Agricultural Sciences, Vienna, Austria.
Jungbauer
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D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide