Immunoelectron microscopy antibodies

Many specific parts of ribosomal RNA molecules and specific proteins within the intact ribosome were located prior to the determination of high resolution crystal structures. One major approach was the use of immunoelectron microscopy. Antibodies to specific ribosomal proteins or to special sites in the RNA were prepared, and electron microscopy was used to map the binding sites of the antibodies on the ribosomal... 

The identity of TES-32 and CTL-1 was confirmed by polyclonal antibodies to recombinant CTL-1, which bound to native TES-32, and by monoclonal antibody Tcn-3, raised to native TES-32 (Maizels et al, 1987), which specifically recognized recombinant CTL-1. The CTL-1 sequence also contained three sites for Afglycosylation, which had previously been shown to be present on TES-32 (Page and Maizels, 1992). Both Tcn-3 and polyclonal antibody to the recombinant CTL-1 protein localize to the cuticle of the infective larvae by immunoelectron microscopy (Fig. 12.2). 

When the light microscope (LM) alone is not sufficient to study a system, the binding of an electron-dense ligand to an antibody facilitates the use of an EM. There are times when, in addition to EM study, immunoelectron microscopic analysis is necessary. Observed anatomical changes often lead to inquiries on the molecular events. This proved true in the case of the report by Inada et al. (22) in which they described a three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles. Immunoelectron microscopy was used to determine the behavior of cellular DNA during senescence. The procedure employed by the investigators is detailed below. 

The peptidyltransferase site. The position was located by binding of derivatives of the antibiotic puromycin (Fig. 29-13). An arylazide derivative of puromycin was photochemically linked (Eq. 23-27) to proteins L23, L18/22, and L15 immunoelectron microscopy, using antibodies to the -dimethyl-adenosine of puromycin,165 166 located the binding site adjacent to the central protuberance between the 50S subunit and 30S subunit near S14.5 4-Thio-dT-p-C-p-puromycin was photochemically crosslinked to G2553... 

An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out (Zaviacic et al., 1994). Immunoelectron microscopy in conjunction with the protein A-gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below. 

Furst, D. O., Osborn, M., and Nave, R. (1988). The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy A map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J. Cell. Biol. 106, 1563-1572. 

Itoh, Y., Suzuki, T., and Kimura, S. (1988). Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies. / Biochem. (Tokyo) 104, 504-508. 

Immunoelectron microscopy (IEM) is the term generally used for techniques that detect the specific binding of antibody to antigen that can be visualized by electron microscopy (1). The use of these techniques results in a 2- to 10,000-fold increase in particle numbers in comparison with methods not using antisera (1) and allows the transmission electron microscope (TEM) to become a sensitive tool, which the plant virus diagnostician or researcher can use to... 

Thouvenin, E., Laurent, S., Madelaine, M.-F., Rasschaert, D., Vautherot, J.-F., and Hewat, E. A. (1997). Bivalent binding of a neutralizing antibody to a calicivirus involves the torsional flexibility of the antibody hinge. / Mol. Biol. 270, 238-246. Roux, K. R. (1984). Direct demonstration of multiple VH allotopes on rabbit Ig molecules Allotope characteristics and Fab arm rotational flexibility revealed by immunoelectron microscopy. Eur. J. Immunol. 14, 459-464. 

Proteins can be detected and quantitated by highly specific antibodies monoclonal antibodies are especially useful because they are homogeneous. Enzyme-linked immunosorbent assays and Western blots of SDS-polyacrylamide gels are used extensively. Proteins can also be localized within cells by immunofluorescence microscopy and immunoelectron microscopy. 

A 67-year-old man presented with an acute bullous eruption 6 weeks after starting bumetanide. He had numerous large tense bullae on erythematous skin, with superficial ulceration on the thighs, arms, and anterior trunk. Pruritus was severe. Routine laboratory tests were normal, except for blood eosinophilia. Biopsy of a blister showed subepidermal bullae associated with dermal infiltrates of neutrophils and eosinophils. Direct immunofluorescence showed continuous linear deposits of C3 and IgG at the basement membrane zone, confirmed by immunoelectron microscopy. Circulating IgG antibasement membrane antibodies were localized in the roof of the blister. Compete clinical heahng and normalization of immunology occurred within 2 months of withdrawal of bumetanide. 

Falkenberg FW, Mondorf U, Pierard D, Ganhl C, Mondorf AW, Mai U, Kantwerk G, Meier U, Rindhage A, Rohracker M. Identification of fragments of proximal and distal tubular cells in the urine of patients under cytostatic treatment by immunoelectron microscopy with monoclonal antibodies. Am J Kidney Dis 1987 9(2) 129-37. 

Immunoelectron microscopy of cardiac muscle revealed that immunogold was deposited on the mitochondria among the myofibrils as well as on the mitochondria beneath the sarcoplasmic membrane (Fig. 20, upper panel). Some immunogold was also seen on the secondary lysosomes and on lipofuscin, which contained heterogeneous substances and showed irregular shapes. In addition, the mitochondria of the endothelial cells of the blood capillaries reacted with this antibody (Fig. 20, lower panel). 

One family of IFAPs, the plakins, is responsible for linking IFs with both microtubules and mlcrofllaments. One plakln family member is plectin, a 500,000-MW protein that has been shown to cross-link intermediate filaments with microtubules and actln filaments in vitro. Plectin also Interacts with other cytoskeletal proteins, including spectrin, microtubule-associated proteins, and lamin B. Immunoelectron microscopy reveals gold-labeled antibodies to plectin decorating short, thin connections between microtubules and vimentin. 

FIGURE 4.39 Immunoelectron microscopy. The opaque particles (150-A, or 15-nm, diameter) in this electron micrograph are clusters of gold atoms bound to antibody molecules. These membrane vesicles from the synapses of neurons contain a channel protein that is recognized by the specific antibody. [Courtesy of Dr. Peter Sargent.]... 

In the rat, the monoclonal antibody MEP-1 stained all cells lining the alveolar space, except the type II pneumocytes (Kasper et al. 1996). Double fluorescence staining employing type I cell-specific lectin BPA (Kasper et al. 1994) revealed the type I cell specificity. Immunoelectron microscopy confirmed this selective reaction of the MEP-1 antibody. The polyclonal anti pan-cathedrin antibody selectively decorated type I pneumocytes, alveolar macrophages and endothelial cells of large blood vessels. Caveolin is a selective marker of type I pneumocytes (Kasper and Reimann 1997). 

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