A modular design of low-background bioassays based on a high-affinity molecular pair barstar:barnase
| Autor: | Ekaterina A. Grebenik, Timothy A. Kelf, Oleg A. Stremovskiy, Varun K. A. Sreenivasan, Jana M. Say, Sergey M. Deyev, Andrei V. Zvyagin, James R. Rabeau |
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| Rok vydání: | 2012 |
| Předmět: |
Proteomics
Receptor ErbB-2 Protein Array Analysis Biotin Biochemistry Antibodies chemistry.chemical_compound Ribonucleases Bacterial Proteins Escherichia coli Molecular Biology Barnase biology Chemistry Avidin Dissociation constant Microscopy Fluorescence Biotinylation biology.protein Biological Assay Barstar Streptavidin Molecular probe Linker |
| Zdroj: | Proteomics. 13(9) |
| ISSN: | 1615-9861 |
| Popis: | High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K(D)) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (K(D) ∼ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (K(D) ∼ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays. |
| Databáze: | OpenAIRE |
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