Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution.
The FLAG epitope is an eight amino acid peptide (AspTyrLysAspAspAspAspLys) that is useful for immunoaffinity purification of fusion proteins. A monoclonal antibody (anti-FLAG M1) that binds the FLAG epitope in a calcium-dependent manner and requires an N-terminal FLAG sequence has been described previously. We describe the use of a second anti-FLAG monoclonal antibody (anti-FLAG M2) in immunoaffinity purification of N-terminal Met-FLAG and C-terminal FLAG fusion to bacterial alkaline phosphatase. Although binding of an anti-FLAG M2 monoclonal antibody to the FLAG epitope is not calcium-dependent, bound fusion proteins can be eluted by competition with FLAG peptide.
Brizzard
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BL
Eastman Kodak Company, New Haven, CT.
Chubet
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Vizard
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DL
D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D000469:Alkaline Phosphatase; D002118:Calcium