A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins*S
By U. Rothbauer, Kourosh Zolghadr, S. Muyldermans, A. Schepers, M. Cardoso, H. Leonhardt. Molecular & Cellular Proteomics, 2008
707 citations recorded by the source corpus.
Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.
Subfields: Biophysics, Molecular Biology, Radiology, Nuclear Medicine and Imaging
View Paper: https://www.semanticscholar.org/paper/dca7f8000486956c4d8bee2ae9b0c57b4b021500
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Citations in the corpus
- Nanobodies: natural single-domain antibodies. (2013, methodology): “This was shown by developing the nanotrap, an immobilized Nb that recognizes the GFP as a target (115), and by Nbs directed against various immunoglobulin isotypes (127).”
- Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. (2012, neutral): “affinity and specificity single-chain antibody from Lama paca (Rothbauer et al., 2008).”
- Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division (2014, methodology): “Total protein extracts were isolated and used for purification with the GFP-TRAP.”