Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.
The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.
Hirsch
James D
JD
Molecular Probes, Inc., Eugene, OR 97402, USA. jim.hirsch@probes.com
Eslamizar
Leila
L
Filanoski
Brian J
BJ
Malekzadeh
Nabi
N
Haugland
Rosaria P
RP
Beechem
Joseph M
JM
Haugland
Richard P
RP
D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007202:Indicators and Reagents; D008024:Ligands; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D019809:Streptavidin / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D002352:Carrier Proteins; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008024:Ligands; D011994:Recombinant Proteins; D014404:Tubulin; C017071:biotin-binding proteins; D001360:Avidin; D001710:Biotin; C004749:desthiobiotin; D012685:Sepharose; D019809:Streptavidin