Generic method for quantification of FLAG-tagged fusion proteins by a real time biosensor.
Availability of rapid quantitative protein-expression analysis is often the bottleneck in high throughput screening applications. A real time biosensor was employed to establish a quantitative assay for FLAG fusion proteins using FLAG-tagged bacterial alkaline phosphatase as standard. A range of FLAG-tagged bacterial alkaline phosphatase concentrations were injected over the anti-FLAG M2 antibody surface of the biosensor and used as standards to determine the concentration of different FLAG-tagged proteins with a molecular mass of 18.1 kDa respectively 49.3 kDa from yeast culture supernatants. The M2 immobilized chip was found to retain binding capacity following regeneration for at least 120 cycles. This real time biosensor method allows the quantitation of proteins from culture supernatants using a calibration curve obtained with a different protein. Further benefits include the short assay time of approximately 5 min, the small amount of sample required (35 microl per injection) and the ability to monitor the binding event in real time.
Mersich
Christa
C
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, Vienna, Austria.
Jungbauer
Alois
A
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D004800:Enzymes, Immobilized / Q000378:metabolism; D005819:Genetic Markers; D020411:Oligonucleotide Array Sequence Analysis; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D005819:Genetic Markers; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D000469:Alkaline Phosphatase