Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation.
AuthorsHwang LC, Gösch M, Lasser T, Wohland T
JournalBiophys J
PubMed ID16632502
'Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. ... More
Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives.
AuthorsEbner A, Marek M, Kaiser K, Kada G, Hahn CD, Lackner B, Gruber HJ,
JournalMethods Mol Biol
PubMed ID18287651
'Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 microM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to ... More
Patterning enzymes inside microfluidic channels via photoattachment chemistry.
AuthorsHolden MA, Jung SY, Cremer PS
JournalAnal Chem
PubMed ID15053641
'We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, ... More
Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization.
AuthorsVasiliskov AV, Timofeev EN, Surzhikov SA, Drobyshev AL, Shick VV, Mirzabekov AD
JournalBiotechniques
PubMed ID10489618
'The manufacturing of microchips containing oligonucleotides and proteins immobilized within gel pads, ranging in size from 10 x 10 to 100 x 100 microns, is described. The microchips are produced by photo- or persulfate-induced copolymerization of unsaturated derivatives of biomolecules with acrylamide-bisacrylamide mixture. Oligonucleotides containing 5''-allyl or 5''-butenediol units were ... More
A streptavidin variant with slower biotin dissociation and increased mechanostability.
AuthorsChivers CE, Crozat E, Chu C, Moy VT, Sherratt DJ, Howarth M,
JournalNat Methods
PubMed ID20383133
'Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important ... More
Biomolecular recognition on well-characterized beads packed in microfluidic channels.
'We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer ... More
Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for biotin-binding proteins.
AuthorsHumbert N, Zocchi A, Ward TR
JournalElectrophoresis
PubMed ID15624156
The biotin-binding protein streptavidin exhibits a high stability against thermal denaturation, especially when complexed to biotin. Herein we show that, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), streptavidin is stabilized at high temperature in the presence of biotinylated fluorescent probes, such as biotin-4-fluorescein, which is incorporated within the binding pocket. ... More
Site-dependent excited-state dynamics of a fluorescent probe bound to avidin and streptavidin.
AuthorsFürstenberg A, Kel O, Gradinaru J, Ward TR, Emery D, Bollot G, Mareda J, Vauthey E,
JournalChemphyschem
PubMed ID19565577
The excited-state dynamics of biotin-spacer-Lucifer-Yellow (LY) constructs bound to avidin (Avi) and streptavidin (Sav) was investigated using femtosecond spectroscopy. Two different locations in the proteins, identified by molecular dynamics simulations of Sav, namely the entrance of the binding pocket and the protein surface, were probed by varying the length of ... More
Impact of hapten presentation on antibody binding at lipid membrane interfaces.
AuthorsJung H, Yang T, Lasagna MD, Shi J, Reinhart GD, Cremer PS,
JournalBiophys J
PubMed ID18199665
We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K(D)) were compared for two model systems, biotin/anti-biotin and ... More
Preservation of the biofunctionality of DNA and protein during microfabrication.
AuthorsTrau D, Jiang J, Sucher NJ
JournalLangmuir
PubMed ID16430241
Microfabrication processes, especially in silicon, are not compatible with biomolecules. Silicon and metal-based materials having crystalline structures are manipulated under harsh conditions with acids, bases, and organic solvents at high temperature. In comparison, organic biomolecules such as DNA and proteins have complex, three-dimensional structures and are sensitive to denaturation, oxidation, ... More
Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein.
AuthorsAslan FM, Yu Y, Mohr SC, Cantor CR
JournalProc Natl Acad Sci U S A
PubMed ID15939877
Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, ... More
Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin.
This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. ... More
Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres.
BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled ... More
Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization.
AuthorsKada G, Kaiser K, Falk H, Gruber HJ
JournalBiochim Biophys Acta
PubMed ID10082986
A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In ... More
Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate.
AuthorsKada G, Falk H, Gruber HJ
JournalBiochim Biophys Acta
PubMed ID10082985
A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand ... More
A streptavidin-biotin binding system that minimizes blocking by endogenous biotin.
Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be ... More