PMID: 6413513Oct 1, 1983Paper

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

The Journal of Cell Biology
J C TalianR D Goldman

Abstract

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.

References

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Citations

Aug 26, 1998·Cell Motility and the Cytoskeleton·R E Stephens, N A Lemieux
Apr 27, 2002·Cell Motility and the Cytoskeleton·Lai-Wa Tam, Paul A Lefebvre
Jul 13, 2002·International Journal of Cancer. Journal International Du Cancer·Albert S MellickNigel A Morrison
May 1, 1994·Molecular Reproduction and Development·L ChristmannR M Moor
Jul 1, 1994·Histochemistry·T Kinoshita, F Sasaki
Apr 28, 2005·Histochemistry and Cell Biology·Jesús EspadaJuan C Stockert
May 12, 1989·Journal of Immunological Methods·D I Stott
May 1, 1985·The Journal of Cell Biology·J M Rifkin, G E Olson
Oct 1, 1987·The Journal of Cell Biology·M W Clark, J Abelson
Nov 1, 1987·The Journal of Cell Biology·J SarasteM G Farquhar
Jan 1, 1988·The Journal of Cell Biology·S P Marchese-RagonaK A Johnson
Apr 1, 1988·The Journal of Cell Biology·B W NeighborsJ R McIntosh
Aug 1, 1988·The Journal of Cell Biology·A M BelkinV E Koteliansky
Aug 15, 2008·Molecular Biology of the Cell·Mark J SchneiderRoger D Sloboda
Jan 1, 1991·Annals of the New York Academy of Sciences·R J Oko, Y Clermont

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