tetramethylrhodamine and pyrene

High concentrations of Tris are effective in dissociating actin-containing complexes, such as the red cell membrane cytoskeleton. A preparative procedure for red cell actin is based on the dissociation of the membrane skeletal complex in a buffer containing 1 M Tris hydrochloride, followed by gel filtration chromatography in the same medium. The actin is recovered as the monomer and is fully native, as judged by its critical concentration of polymerization, inhibition of DNase I, stimulation of myosin ATPase, and the appearance in the electron microscope of filaments, both bare and decorated with heavy meromyosin, and of magnesium ion-induced paracrystals. The Tris solution causes rapid depolymerization of F-actin with no denaturation, and the solution of monomeric actin in this medium is stable for many weeks in the cold; concentrated Tris is more reliable than guanidinium chloride for the depolymerization of F-actin in the estimation of total actin concentration by the DNase I inhibition assay.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biophysical Phenomena; Biophysics; Cations, Divalent; Chromatography; Cytoskeleton; Deoxyribonuclease I; DNA; Erythrocytes; Fluorometry; Guanidine; Guanidines; Humans; In Vitro Techniques; Microscopy, Electron; Muscle, Skeletal; Myosin Subfragments; Nucleotides; Phalloidine; Polymers; Pyrenes; Rabbits; Rhodamines; Tromethamine

A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and dansyl, were evaluated for this purpose. Also, this technology is ideal for fluorescence energy-transfer immunoassays that require labeling of the antibody with either a donor or acceptor fluorophore. In such cases, a fluorescent dye can be specifically attached near the antigen-combining site, where it can participate in high-efficiency energy transfer with its complementary fluorophore attached to the antigen.

Topics: Affinity Labels; Antibodies; Biotin; Dansyl Compounds; Fluorescent Dyes; Fluoroimmunoassay; Molecular Structure; Pyrenes; Rhodamines