Development of a new and simple method for the detection of histidine-tagged proteins

To develop a general method for the detection of histidine-tagged proteins, the interactions of the histidine epitope tag of MutH and MutL proteins with the epitope specific monoclonal anti-His(6) antibody were monitored by a label-free direct method using impedance spectroscopy. The immumosensor was fabricated by covalent coupling of the antibody on a conducting polymer coated electrode surface. The impedance of the antibody modified electrode was decreased after binding to the histidine-tagged proteins. The specificity of the sensor was demonstrated by showing that no impedance change was occurred when the sensor was exposed to both of non-tagged MutH and MutL proteins. The specific interaction was further characterized using quartz crystal microbalance studies. Based on impedance measurements, the linear ranges were obtained from 50.0 to 125.0 and 50.0 to 250.0 mug/ml, for His-tag MutH and His-tag MutL proteins, respectively. The detection limits were determined to be 37.8 and 59.1 mug/ml, for His-tag MutH and His-tag MutL proteins, respectively. (C) 2004 Elsevier B.V. All rights reserved.