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-His6 antibody were monitored by a label-free direct method using impedance spectroscopy. The immunosensor 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 micorg/ml, for His-tag MutH and His-tag MutL proteins, respectively. The detection limits were determined to be 37.8 and 59.1 microg/ml, for His-tag MutH and His-tag MutL proteins, respectively.
Darain
Farzana
F
Department of Chemistry and Center for Innovative Bio-Physio Sensor Technology, Pusan National University, Pusan 609-735, South Korea.
Ban
Changill
C
Shim
Yoon-Bo
YB
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D045643:DNA Repair Enzymes / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D017097:Electric Impedance; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D006639:Histidine / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D007118:Immunoassay / Q000379:methods*; D000070956:MutL Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes