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. (ii) Fluorescent biotin derivatives were synthesized by derivatizing e...

Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of ...

1. The optical-rotatory-dispersion and circular-dichroism curves of avidin showed positive Cotton effects centred at 228mmu and 280mmu, close to the ultraviolet-absorption bands of tryptophan. These effects disappeared when avidin was dissociated into sub-units in guanidine hydrochloride. 2. Binding of biotin had only a small effect on the optical-rotatory-dispersion curve of avidin. 3. The abs...

To measure the depth of the local anesthetic binding site within the neuronal membrane, biotin-containing polyethylene glycols having zero, three, and six ethylene glycol subunits were added to the p-amino termini of tetracaine and procaine, thereby interposing a pharmacologically inert "spacer" molecule between the local anesthetic and the biotin moiety. These biotinyl-local anesthetic derivat...

Avidin, which is one type of glycoprotein, has a strong affinity with biotin (Ka = 10(15) M(-1)). Iminobiotin also forms a complex with avidin (Ka = 10(8) M(-1) at pH 9.5). The avidin-iminobiotin complex changes to the avidin-biotin complex in the presence of biotin because of the difference of the binding constant to avidin. In this study, the interaction between avidin and iminobiotin labeled...

The biotin switch assay for detection of protein S-nitrosation has been widely used in the field of nitric oxide and redox signaling. However, here we found that there is experimental and theoretical interference of intermolecular disulfide bonds in S-nitrosated protein identification with avidin purification after biotin switch method: proteins linked to S-nitrosated proteins by intermolecular...

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biot...

Coadsorption of high-affinity avidin with lower affinity cell adhesion protein fibronectin has been shown to significantly augment short-term (1 h) adhesion and spreading of endothelial cells; however, the longer term persistence of avidin binding and its effect on endothelial cell adhesion have not been addressed. In this study, the presence of avidin-biotin bonds 24 h after cell adhesion to t...

PURPOSE The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells. METHODS Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4 degrees C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex ...