Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein.

Abstract:

:Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Oberbichler E,Wiesauer M,Schlögl E,Stangl J,Faschinger F,Knör G,Gruber HJ,Hytönen VP

doi

10.1016/bs.mie.2019.10.028

subject

Has Abstract

pub_date

2020-01-01 00:00:00

pages

1-20

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(19)30441-0

journal_volume

633

pub_type

杂志文章
  • Applications of Dissolution-DNP for NMR Screening.

    abstract::Experimental screening for protein-ligand interactions is a central task in drug discovery. Nuclear magnetic resonance (NMR) spectroscopy enables the determination of binding affinities, as well as the measurement of structural and dynamic parameters governing the interaction. With traditional liquid-state NMR relying...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.08.016

    authors: Kim Y,Hilty C

    更新日期:2019-01-01 00:00:00

  • Purification and Structural Analysis of Desmoplakin.

    abstract::Desmoplakin (DP) is an obligate component of desmosomes, where it links the desmosomal cadherin/plakoglobin/plakophilin assembly to intermediate filaments. DP contains a large amino-terminal domain (DPNT) that binds to the cadherin/plakoglobin/plakophilin complex, a central coiled-coil domain that dimerizes the molecu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.05.006

    authors: Choi HJ,Weis WI

    更新日期:2016-01-01 00:00:00

  • Roles of GlcNAc-6-O-sulfotransferases in lymphoid and nonlymphoid tissues.

    abstract::Recent studies using sulfotransferase-deficient mice have revealed various physiological functions of sulfated glycans. Studies using gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 showed that these sulfotransferases play critical roles in lymphocyte homing....

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)79014-5

    authors: Kawashima H

    更新日期:2010-01-01 00:00:00

  • Inhibitors of advanced glycation and endoplasmic reticulum stress.

    abstract::Advanced glycation is one of the major pathophysiological posttranslational modifications. Under hyperglycemic conditions or oxidative stress, proteins and DNA are nonenzymatically modified by oxidative glycation and converted to advanced glycation endproducts (AGEs), which induce the loss of protein functions or apop...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385928-0.00020-1

    authors: Inagi R

    更新日期:2011-01-01 00:00:00

  • Expression of Cellulolytic Enzyme as a Fusion Protein That Reacts Specifically With a Polymeric Scaffold.

    abstract::The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes buil...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.12.003

    authors: Katyal P,Yang Y,Vinogradova O,Lin Y

    更新日期:2017-01-01 00:00:00

  • Manipulation of PKC isozymes by RNA interference and inducible expression of PKC constructs.

    abstract::Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in apoptosis. Several members of the PKC family act as substrates for caspases. In addition, PKCs can also regulate caspase activation and cell death by apoptosis. The cleavage of PKCs separates the regulatory domain from the catalyt...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)01608-X

    authors: Basu A,Persaud SD,Sivaprasad U

    更新日期:2008-01-01 00:00:00

  • Metal-free chromatographic media.

    abstract::The three major considerations in reducing metal-gel binding are (1) the type of gel to be used, (2) the ionic strength of the eluent, and (3) alkaline reduction of the gel. Each of these parameters should be considered in advance of an experiment. A method is offered for determining the optimal gel type, gel treatmen...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(88)58044-8

    authors: Martin MT

    更新日期:1988-01-01 00:00:00

  • Functional Characterization of Histone Chaperones Using SNAP-Tag-Based Imaging to Assess De Novo Histone Deposition.

    abstract::Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specifici...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.04.004

    authors: Clément C,Vassias I,Ray-Gallet D,Almouzni G

    更新日期:2016-01-01 00:00:00

  • Sensitive detection of structural features and rearrangements in long, structured RNA molecules.

    abstract::Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suite...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.04.002

    authors: Adams RL,Huston NC,Tavares RCA,Pyle AM

    更新日期:2019-01-01 00:00:00

  • Nuclear magnetic resonance measurements of slow conformational dynamics in macromolecules.

    abstract::The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constant...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/s0076-6879(94)39023-1

    authors: Lane AN,Lefèvre JF

    更新日期:1994-01-01 00:00:00

  • Characterization of oligophrenin-1, a RhoGAP lost in patients affected with mental retardation: lentiviral injection in organotypic brain slice cultures.

    abstract::Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein, was one of the first Rho-linked MR genes identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)00419-3

    authors: Kasri NN,Govek EE,Van Aelst L

    更新日期:2008-01-01 00:00:00

  • Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.

    abstract::There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)00409-0

    authors: Onesto C,Shutes A,Picard V,Schweighoffer F,Der CJ

    更新日期:2008-01-01 00:00:00

  • Use of acoustic sensors to probe the mechanical properties of liposomes.

    abstract::Acoustic sensors probe the response of a thin layer to the mechanical displacement associated with an acoustic wave. Acoustic measurements provide two simultaneous time-resolved signals; one signal is related to the velocity or frequency of the acoustic wave and is mainly a function of adsorbed mass, while the second ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)65002-3

    authors: Melzak K,Tsortos A,Gizeli E

    更新日期:2009-01-01 00:00:00

  • Application of Monte Carlo-based receptor ensemble docking to virtual screening for GPCR ligands.

    abstract::Receptor ensemble docking (RED) is an effective strategy to account for receptor flexibility in the course of a docking-based virtual screening campaign. Such an approach can be applied when multiple crystal structures of a receptor have been solved, but it can also be applied when only a single crystal structure is a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-407865-9.00014-5

    authors: Vilar S,Costanzi S

    更新日期:2013-01-01 00:00:00

  • TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

    abstract::Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1. ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.04.024

    authors: Young AP,Bandarian V

    更新日期:2018-01-01 00:00:00

  • Examining histone posttranslational modification patterns by high-resolution mass spectrometry.

    abstract::Histone variants and posttranslational modifications (PTMs) are essential for epigenetic regulation of transcriptional expression. Single and/or combinatorial PTMs of histones play important roles in development and disease formation. Mass spectrometry (MS) has been a powerful tool to study histone variants and PTMs, ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-391940-3.00001-9

    authors: Lin S,Garcia BA

    更新日期:2012-01-01 00:00:00

  • Analysis of polysomes from bacteria.

    abstract::In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate fast...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-420037-1.00008-7

    authors: Qin D,Fredrick K

    更新日期:2013-01-01 00:00:00

  • Functional Study of the Vitamin K Cycle Enzymes in Live Cells.

    abstract::Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylati...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.10.015

    authors: Tie JK,Stafford DW

    更新日期:2017-01-01 00:00:00

  • Designer cellulosomes for enhanced hydrolysis of cellulosic substrates.

    abstract::During the past several years, major progress has been accomplished in the production of "designer cellulosomes," artificial enzymatic complexes that were demonstrated to efficiently degrade crystalline cellulose. This progress is part of a global attempt to promote biomass waste solutions and biofuel production. In d...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-415931-0.00023-9

    authors: Vazana Y,Moraïs S,Barak Y,Lamed R,Bayer EA

    更新日期:2012-01-01 00:00:00

  • Quantitative determination of phagocytosis by bone marrow-derived dendritic cells via imaging flow cytometry.

    abstract::Immunogenic cell death (ICD), induced by certain anticancer chemotherapeutics, leads to the emission of danger associated molecular patterns (DAMP) by cancer cells, which facilitates the attraction, activation and maturation of dendritic cells (DC) as well as the subsequent priming of effector T cells. In this context...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.07.021

    authors: Cerrato G,Liu P,Martins I,Kepp O,Kroemer G

    更新日期:2020-01-01 00:00:00

  • Mammalian photoentrainment: results, methods, and approaches.

    abstract::Research on circadian biology over the past decade has paid increasing attention to the photoreceptor mechanisms that align the molecular clock to the 24-h light/dark cycle, and some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for entrainment. A po...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(05)93037-1

    authors: Peirson SN,Thompson S,Hankins MW,Foster RG

    更新日期:2005-01-01 00:00:00

  • trLRET microscopy: Ultrasensitive imaging of lanthanide luminophores.

    abstract::In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limit...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.04.030

    authors: Ciepla P,Cho U,Chen JK

    更新日期:2020-01-01 00:00:00

  • Profiling DUBs and Ubl-specific proteases with activity-based probes.

    abstract::Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.12.037

    authors: Geurink PP,van der Heden van Noort GJ,Mulder MPC,Knaap RCM,Kikkert M,Ovaa H

    更新日期:2019-01-01 00:00:00

  • Mass mapping of large globin complexes by scanning transmission electron microscopy.

    abstract::Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the project...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)36027-3

    authors: Wall JS,Simon MN,Lin BY,Vinogradov SN

    更新日期:2008-01-01 00:00:00

  • Fe-S Cluster Hsp70 Chaperones: The ATPase Cycle and Protein Interactions.

    abstract::Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/bs.mie.2017.07.004

    authors: Dutkiewicz R,Nowak M,Craig EA,Marszalek J

    更新日期:2017-01-01 00:00:00

  • The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.

    abstract::In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)09019-1

    authors: Storici F,Resnick MA

    更新日期:2006-01-01 00:00:00

  • Phosphotransfer profiling: systematic mapping of two-component signal transduction pathways and phosphorelays.

    abstract::Two-component signal transduction systems, composed of histidine kinases and response regulators, enable bacteria to sense, respond, and adapt to changes in their internal and external conditions. The importance of these signaling systems is reflected in their widespread distribution and prevalence in the bacterial ki...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)23026-5

    authors: Laub MT,Biondi EG,Skerker JM

    更新日期:2007-01-01 00:00:00

  • Measuring synchrony in the mammalian central circadian circuit.

    abstract::Circadian clocks control daily rhythms in physiology and behavior across all phyla. These rhythms are intrinsic to individual cells that must synchronize to their environment and to each other to anticipate daily events. Recent advances in recording from large numbers of cells for many circadian cycles have enabled re...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/bs.mie.2014.10.042

    authors: Herzog ED,Kiss IZ,Mazuski C

    更新日期:2015-01-01 00:00:00

  • Tubulin as a regulator of G-protein signaling.

    abstract::Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses me...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(04)90024-9

    authors: Rasenick MM,Donati RJ,Popova JS,Yu JZ

    更新日期:2004-01-01 00:00:00

  • The role of EFA6, exchange factor for Arf6, for tight junction assembly, functions, and interaction with the actin cytoskeleton.

    abstract::In polarized epithelial cells, the tight junction has been ascribed several functions including the regulation of the paracellular permeability, an impediment to the diffusion of molecules between the apical and basolateral domains, a site of delivery of transport vesicles for basolateral proteins, and a scaffold for ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)04029-2

    authors: Luton F

    更新日期:2005-01-01 00:00:00