scholarly journals Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

2014 ◽  
Vol 406 ◽  
pp. 143-147 ◽  
Author(s):  
Raju Ahmed ◽  
Emma Spikings ◽  
Shaobo Zhou ◽  
Andrew Thompsett ◽  
Tiantian Zhang
Keyword(s):  
Detection System ◽  
Binding Activity ◽  
Biotin Binding ◽  
Endogenous Biotin

scholarly journals Biotin binding to avidin. Oligosaccharide side chain not required for ligand association

1987 ◽  
Vol 248 (1) ◽  
pp. 167-171 ◽  
Author(s):  
Y Hiller ◽  
J M Gershoni ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Concanavalin A ◽  
Sugar Content ◽  
Binding Activity ◽  
Side Chain ◽  
Affinity Column ◽  
Binding Properties ◽  
Commercial Preparation ◽  
Oligosaccharide Moiety ◽  
Biotin Binding

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli

2018 ◽  
Vol 40 (3) ◽  
pp. 591-600
Author(s):  
H. D. Pardavé-Alejandre ◽  
J. E. Alvarado-Yaah ◽  
E. N. Pompa-Mera ◽  
J. E. Muñoz-Medina ◽  
B. Sárquiz-Martínez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Activity ◽  
Biotin Binding

Novel application of streptavidin-hapten derivatives as protein-tracer conjugate in competitive-type immunoassays involving biotinylated detection probes

1991 ◽  
Vol 37 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton
Keyword(s):  
No Reduction ◽  
Solid Phase ◽  
Binding Activity ◽  
Fluorescence Signal ◽  
Antibody Immobilization ◽  
Aggregate Formation ◽  
Cross Link ◽  
Time Resolved ◽  
Biotin Binding ◽  
Detection Reagent

Abstract To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

Immunohistochemical study of the distribution of endogenous biotin and biotin-binding enzymes in ductal structures of salivary gland tumours

2000 ◽  
Vol 29 (9) ◽  
pp. 445-451 ◽  
Author(s):  
Chang-Sheng Lu ◽  
Kenji Kashima ◽  
Tsutomu Daa ◽  
Shigeo Yokoyama ◽  
Shigetaka Yanagisawa ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Immunohistochemical Study ◽  
Salivary Gland Tumours ◽  
Biotin Binding ◽  
Endogenous Biotin

scholarly journals Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site

1988 ◽  
Vol 256 (1) ◽  
pp. 279-282 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Reversed Phase ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Biotin Binding ◽  
Peptide Fractions ◽  
Specific Reagent

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

scholarly journals High Resolution Screening of Plant Natural Product Extracts for Estrogen Receptor a and f3 Binding Activity Using an Online HPLC-MS Biochemical Detection System

2001 ◽  
Vol 6 (5) ◽  
pp. 291-303 ◽  
Author(s):  
Uwe Schobel ◽  
Michel Frenay ◽  
Danny A. Van Elswijk ◽  
Joanne M. McAndrews ◽  
Kelly R. Long ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
High Resolution ◽  
Natural Product ◽  
High Speed ◽  
Detection System ◽  
Binding Activity ◽  
Biologically Active ◽  
Fluorescence Detector ◽  
Active Compounds ◽  
Activation Assay

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

Detection of Marine Toxins Using Reconstituted Sodium Channels

1995 ◽  
Vol 78 (2) ◽  
pp. 570-573 ◽  
Author(s):  
Vera L Trainer ◽  
Daniel G Baden ◽  
William A Catterall
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Biochemical Analysis ◽  
Detection System ◽  
Specific Binding ◽  
Binding Activity ◽  
Phospholipid Vesicles ◽  
Marine Toxins ◽  
Receptor Sites ◽  
Experimental Preparation

Abstract Specific binding of the marine toxins saxitoxin, tetrodotoxin, and brevetoxin to the rat brain sodium channel is demonstrated using purified sodium channels reconstituted into phospholipid vesicles. Restoration of sodium channel function and binding activity by incorporation into phospholipid vesicles provides the only rigorous proof that the purified protein contains the neurotoxin receptor sites. In addition, reconstitution provides a valuable experimental preparation for biochemical analysis of neurotoxin binding sites and may facilitate the development of a specific toxin detection system.

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