Site-Specific Immobilization of Biotinylated Proteins for Protein Microarray Analysis

Abstract

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.

Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas

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A Simple Parallel Analytical Method of Prenatal Screening

“…The advance in protein microarray technology has benefited the medical community greatly, making the parallel analysis of multiple correlative biomarkers a reality [1,2] . In the past few years, researchers all over the world have developed a number of protein microarray products for use in diagnosis, prognosis, prevention and therapy [3][4][5][6][7][8] .…”

Incorporation of non-natural modules into proteins: structural features beyond the genetic code

“…Immobilization proceeded selectively via the azido group and the immobilized enzyme retained its catalytic activity and was able to bind to the ribonuclease inhibitor protein. Alternatively, biotinylation of proteins by EPL enables the oriented immobilization onto avidinfunctionalized supports (Lue et al 2004). As in contrast to conventional immobilization methods the oriented immobilization yields homogenous protein species, this approach is expected to be highly advantageous for the application of immobilized proteins.…”