Wheat germ agglutinin-apoHRP gold: A new retrograde tracer for light- and electron-microscopic single- and double-label studies

1987 ◽  
Vol 261 (2) ◽  
pp. 306-318 ◽  
Author(s):  
A. I. Basbaum ◽  
D. Menetrey
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Electron Microscopic ◽  
Retrograde Tracer ◽  
Double Label

scholarly journals Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

1977 ◽  
Vol 25 (11) ◽  
pp. 1187-1200 ◽  
Author(s):  
W D Geoghegan ◽  
G A Ackerman
Keyword(s):  
Horseradish Peroxidase ◽  
Concanavalin A ◽  
Colloidal Gold ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Indirect Detection ◽  
Electron Microscopic Level ◽  
Specific Cell ◽  
Major Protein ◽  
Electron Microscopic

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.

scholarly journals Subfractionation of cardiac sarcolemma with wheat-germ agglutinin

1989 ◽  
Vol 264 (3) ◽  
pp. 885-892 ◽  
Author(s):  
J H M Charuk ◽  
S Howlett ◽  
M Michalak
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Freeze Fracture ◽  
Protein Composition ◽  
Tubular Structures ◽  
Electron Microscopic ◽  
Cardiac Sarcolemma ◽  
Inside Out

The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.

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