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Cell‐Surface Recognition of Biotinylated Membrane Proteins Requires Very Long Spacer Arms: An Example from Glucose‐Transporter Probes
Author(s) -
Hashimoto Makoto,
Yang Jing,
Holman Geoffrey D.
Publication year - 2001
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20010105)2:1<52::aid-cbic52>3.0.co;2-f
Subject(s) - biotinylation , biotin , streptavidin , chemistry , avidin , membrane , glucose transporter , biochemistry , transporter , adduct , cell membrane , biophysics , combinatorial chemistry , organic chemistry , biology , gene , insulin , endocrinology
Glucose transporters (GLUTs) can be photoaffinity labelled by (diazirinetrifluoroethyl)benzoyl‐substituted glucose derivatives and the adduct can be recognised, after detergent solubilisation of membranes, by using streptavidin‐based detection systems. However, in intact cells recognition of photolabelled GLUTs by avidin and anti‐biotin antibodies only occurs if the bridge between the photoreactive and the biotin moieties has a minimum of 60–70 spacer atoms. We show that a suitably long bridge can be synthesised with a combination of polyethylene glycol and tartarate groups and that introduction of these spacers generates hydrophilic products that can be cleaved with periodate. Introduction of the very long spacers does not appreciably reduce the affinity of interaction of the probes with the transport system.