Site-specific immobilization of biotinylated proteins for protein microarray analysis.
The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide. However, the binding between Ni-NTA and His tag proteins is not strong, causing the immobilized proteins to dissociate from the slide even under simple wash conditions. The authors have developed a novel strategy of using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. Array-scan results not only show successful immobilization of proteins onto slides by antibody detection method but also full retention of biological activities of the immobilized proteins. The strong and specific interaction between biotin and avidin also permits the use of stringent incubation and washing conditions on the protein microchip, thus making it a highly robust method for array studies.
Lue
Rina Y P
RY
Department of Biological Sciences, National University of Singapore.
Chen
Grace Y J
GY
Zhu
Qing
Q
Lesaicherre
Marie-Laure
ML
Yao
Shao Q
SQ
D001360:Avidin / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D011993:Recombinant Fusion Proteins; D001360:Avidin; D001710:Biotin; D003545:Cysteine