Protocol abstract
Nature Protocols 3, - 534 - 545 (2008)
Published online: 6 March 2008 | doi:10.1038/nprot.2008.20
Subject Categories: Biochemistry and protein analysis | Chemical modification | Imaging
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin
Mark Howarth1,2 & Alice Y Ting1
Abstract
This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
- Present address: Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK.
Correspondence to: Mark Howarth1,2 e-mail: mark.howarth@bioch.ox.ac.uk
Correspondence to: Alice Y Ting1 e-mail: ating@mit.edu
nature-products
A-Z product listing
- Alexa Fluor 568 succinimidyl ester(Invitrogen)
- ATP(Sigma)
- B-PER(Pierce)
- Biotin(Sigma)
- Biotin–adenosine monophosphate(Ting lab)
- BirA(Avidity)
MORE ARTICLES LIKE THIS
These links to content published by NPG are automatically generated.
NEWS AND VIEWS
Chimeric streptavidins with reduced valenciesNature Methods News and Views (01 Apr 2006)
