scholarly journals Effect of tissue processing on colloidal gold cytochemistry.

1987 ◽  
Vol 35 (9) ◽  
pp. 983-996 ◽  
Author(s):  
M Bendayan ◽  
A Nanci ◽  
F W Kan
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Protein A ◽  
Biochemical Properties ◽  
Physical Conditions ◽  
Single Procedure ◽  
Cell Compartments ◽  
Biochemical Components ◽  
Structural Preservation ◽  
Observation Results

The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell compartments. However, since preparation of tissues for structural observation results in major alterations of the properties of their components, a major problem is to retain an adequate degree of their biochemical properties as well as adequate structural preservation. In the present study, we describe results obtained using various colloidal gold cytochemical techniques on tissues processed through different approaches. We found that any manipulation of the tissue during its processing can result in modifications of tissue components, leading to problems in cytochemistry. Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions of embedding can all introduce changes in tissue components which can be cytochemically demonstrated. This has been illustrated with application of the protein A-gold, lectin-gold, and enzyme-gold cytochemical techniques on tissues submitted to different processings: fixation by perfusion or by immersion; glutaraldehyde vs paraformaldehyde fixative solutions; cryo-ultramicrotomy; embedding in epoxy, GMA, Lowicryl, or LR resins. The results obtained have demonstrated that conditions for optimal labeling must be worked out for each class of binding sites, and that no single procedure can be recommended as THE best approach in cytochemistry.

Facts and artifacts in colloidal gold postembedding cytochemistry

1986 ◽  
Vol 44 ◽  
pp. 44-47
Author(s):  
Moise Rendayan
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Protein A ◽  
Light And Electron Microscopy ◽  
Ultrastructural Localization ◽  
Surface Antigens ◽  
Enzyme Cytochemistry ◽  
Intracellular Binding ◽  
Gold Marker

The colloidal gold marker was introduced in immunocytochemistry by Faulk and Taylor, in 1971, for the ultrastructural localization of surface antigens. Since then, application of this marker in light and electron microscopy has been growing rapidly. In particular, it has been applied for postembedding labeling of intracellular binding sites and its use has been extended to the various fields of cytochemistry: immunocytochemistry (protein A-gold), IgG-gold), enzyme-cytochemistry and lectincytochemistry. Several reviews have been recently published on colloidal gold labeling techniques and we refer to them for extensive characterization of this marker and its various applications.

scholarly journals Protein AG-gold complex: an alternative probe in immunocytochemistry.

1991 ◽  
Vol 39 (8) ◽  
pp. 1057-1065 ◽  
Author(s):  
L Ghitescu ◽  
Z Galis ◽  
M Bendayan
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Protein A ◽  
Particle Surface ◽  
Surface Protein ◽  
Immunoblot Analysis ◽  
Gold Complex ◽  
Protein G ◽  
Staphylococcal Protein A ◽  
Monoclonal Immunoglobulins

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

scholarly journals Interaction of calcium ions and salivary acidic proline-rich proteins with hydroxyapatite. A possible aspect of inhibition of hydroxyapatite formation

1983 ◽  
Vol 213 (1) ◽  
pp. 11-20 ◽  
Author(s):  
A Bennick ◽  
D Kells ◽  
G Madapallimattam
Keyword(s):  
Crystal Growth ◽  
Calcium Ions ◽  
Binding Sites ◽  
Protein C ◽  
Tryptic Peptide ◽  
Protein A ◽  
Additional Amount ◽  
The Relationship ◽  
Surrounding Liquid ◽  
Hydroxyapatite Formation

The relationship between Ca2+- and hydroxyapatite-binding sites in salivary acidic proline-rich phosphoproteins A and C was investigated. Coating of hydroxyapatite with protein before adsorption had no effect on Ca2+ binding to the mineral, but simultaneous adsorption of Ca+ and protein to hydroxyapatite caused additional Ca2+ binding to the solid. The additional amount of Ca2+ adsorbed, measured in mol of Ca2+/mol of protein adsorbed to hydroxyapatite, was approx. 2 for protein C, 4 for protein A, 9 for the N-terminal tryptic peptide and 2 for dephosphorylated protein A. It is suggested that the ability of the proteins to inhibit hydroxyapatite formation is related to the binding of the proteins to crystal growth sites on the mineral, which prevents access of Ca2+ from the surrounding liquid.

scholarly journals Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus

2003 ◽  
Vol 31 (3) ◽  
pp. 716-718 ◽  
Author(s):  
N.G. Housden ◽  
S. Harrison ◽  
S.E. Roberts ◽  
J.A. Beckingham ◽  
M. Graille ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein A ◽  
Cell Wall Protein ◽  
Protein G ◽  
Protein L ◽  
Heavy Chains ◽  
Binding Domains ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

Protein L is a multidomain cell-wall protein isolated from Peptostreptococcus magnus. It belongs to a group of proteins that contain repeated domains that are able to bind to Igs without stimulating an immune response, the most characterized of this group being Protein A (Staphylococcus aureus) and Protein G (Streptococcus). Both of these proteins bind predominantly to the interface of CH2-CH3 heavy chains, while Protein L binds exclusively to the VL domain of the κ-chain. The function of these proteins in vivo is not clear but it is thought that they enable the bacteria to evade the host's immune system. Two binding sites for κ-chain on a single Ig-binding domain from Protein L have recently been reported and we give evidence that one site has a 25–55-fold higher affinity for κ-chain than the second site.

scholarly journals Effect of Cysteine 85 on Biochemical Properties and Biological Function of Human Surfactant Protein A Variants†

Biochemistry ◽  
2007 ◽  
Vol 46 (28) ◽  
pp. 8425-8435 ◽  
Author(s):  
Guirong Wang ◽  
Catherine Myers ◽  
Anatoly Mikerov ◽  
Joanna Floros
Keyword(s):  
Biological Function ◽  
Protein A ◽  
Surfactant Protein ◽  
Biochemical Properties ◽  
Surfactant Protein A

scholarly journals Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

2021 ◽  
Vol 12 ◽  
Author(s):  
Weizhi Ouyang ◽  
Xiwen Zhang ◽  
Yong Peng ◽  
Qing Zhang ◽  
Zhilin Cao ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Protein A ◽  
Transcriptional Factor ◽  
Experimental Procedure ◽  
Histone Marks ◽  
Genome Wide ◽  
Efficient Alternative ◽  
Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

scholarly journals Preparation of Protein a Gold

1997 ◽  
Vol 5 (5) ◽  
pp. 12-13
Author(s):  
Paul Webster
Keyword(s):  
Electron Microscopy ◽  
Biological Activity ◽  
Colloidal Gold ◽  
Protein A ◽  
List Type ◽  
Type Number ◽  
Stained Glass ◽  
Small Particles ◽  
Silver Enhancement ◽  
Gold Particles

Colloidal gold has been used for centuries in the preparation of stained glass for windows and fine glassware. In recent years, colloidal gold particles have become a useful tool in microscopy for staining tissues and sections. Colloidal gold particles are especially useful for biological electron microscopy, Some of the reasons why are listed below.*Homogeneous preparations of particles varying in size from 3μm to 20μm can be easily prepared.*Colloidal gold suspensions are inexpensive to prepare. Most proteins can be easily coupled to colloidal gold particles.*Most proteins can be easily coupled to colloidal gold particles.*Proteins coupled to gold particles do not appear to lose their biological activity.*The colloidal gold particles can be easily seen in the electron microscope.*Colloidal gold does not naturally occur in biological material. Therefore, if you see it, it is because you put it there.*Colloidal gold probes can be used for light microscopy, The larger gold particles can be directly observed by the light microscope. Small particles are detected by silver enhancement or epipolarized illumination.*The same probes can be used for both LM and TEM imrnunocytochemistry.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

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