scholarly journals R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues.

1991 ◽  
Vol 39 (7) ◽  
pp. 921-926 ◽  
Author(s):  
G Sohn ◽  
C Sautter
Keyword(s):  
Indirect Immunofluorescence ◽  
Binding Sites ◽  
Aquatic Macrophytes ◽  
Elodea Canadensis ◽  
Fluorescent Labeling ◽  
Fluorescent Label ◽  
Nonspecific Binding ◽  
Immunofluorescence Method ◽  
Rabbit Igg ◽  
Highly Sensitive

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Eliminating Nonspecific Binding Sites for Highly Reliable Immunoassay via Super-resolution Multicolor Fluorescence Colocalization

Nanoscale ◽  
2021 ◽  
Author(s):  
Shenfei Zong ◽  
Yun Liu ◽  
Kuo Yang ◽  
Zhaoyan Yang ◽  
Zhuyuan Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Super Resolution ◽  
Specific Adsorption ◽  
Nonspecific Binding ◽  
Nonspecific Adsorption ◽  
Multicolor Fluorescence

Non-specific adsorption in immunoassays has always been a major problem that affects the reliability of assay results. Despite the emergence of various methods which can reduce nonspecific adsorption, a universal...

scholarly journals 3 minutes to precisely measure morphogen concentration

2018 ◽  
Author(s):  
Tanguy Lucas ◽  
Huy Tran ◽  
Carmina Angelica Perez Romero ◽  
Aurélien Guillou ◽  
Cécile Fradin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Response ◽  
Positional Information ◽  
Fruit Fly ◽  
Fluorescent Labeling ◽  
Hill Coefficient ◽  
Activation Process ◽  
Endogenous Expression ◽  
Time Period ◽  
Zygotic Transcription

AbstractMorphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

scholarly journals Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.

1984 ◽  
Vol 98 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Walsh
Keyword(s):  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Indirect Immunofluorescence ◽  
Cell Protein ◽  
Basal Bodies ◽  
Immunofluorescence Method ◽  
Nonionic Detergent ◽  
Cytoplasmic Microtubules ◽  
Spherical Cells ◽  
Naegleria Gruberi

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

Polarization and density of Na-pumps in the inner ear of the chick embryo during early stages of development

Development ◽  
1987 ◽  
Vol 100 (2) ◽  
pp. 271-278
Author(s):  
F. Giraldez ◽  
J.J. Represa ◽  
L. Borondo ◽  
E. Barbosa
Keyword(s):  
Binding Sites ◽  
Fluid Transport ◽  
Developmental Stages ◽  
Otic Vesicle ◽  
Transient Stage ◽  
Highly Sensitive ◽  
Proliferative Growth ◽  
K Concentration ◽  
Otic Vesicles ◽  
Vectorial Transport

The otic vesicle consists of a pseudostratified epithelium with some features of transporting epithelia. The present work questions whether Na-pumps are polarized in this epithelium and what is the relation between the location or density of pumps and development. This was done by measuring the binding of [3H]ouabain to isolated otic vesicles in developmental stages 16 to 22. The results show the presence of specific ouabain-binding sites located in the inward-facing membrane of the otic vesicle epithelium. Binding was saturable at increasing concentrations of ouabain and was highly sensitive to the external K+ concentration with half-maximal inhibition below 0.5 mM, indicating that the binding sites and Na-pump sites are identical. A transient stage-dependent increase in the density of Na-pumps during the period that precedes growth was observed. Evidence is given against this being directly related to an increased net fluid transport rate despite the fact that pump sites were always polarized throughout these stages. The conclusions are that (1) the otic vesicle epithelium is a polarized structure with the possibility of vectorial transport of solutes and water and (2) the increased number of pumps may operate as a regulatory mechanism during normal proliferative growth.

scholarly journals Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

1985 ◽  
Vol 33 (5) ◽  
pp. 474-476 ◽  
Author(s):  
V Muresan ◽  
M C Constantinescu
Keyword(s):  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Binding Sites ◽  
Sodium Periodate ◽  
Luminal Surface ◽  
Nonspecific Binding ◽  
Coated Pits ◽  
Neuraminidase Treatment ◽  
Plasmalemmal Vesicles ◽  
Endothelial Surface

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

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