Ref ID : 4400
Robert Kennie Peck; Specificity of an Antiserum Produced against Pseudomicrothorax dubius Trichocysts Prepared by a Rapid Isolation Method. J.Protozool. 39(1):39-45, 1992
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A rapid method was developed for the isolation of Pseudomicrothorax dubius ciliary and trichocyst fractions which were characterized by SDS-PAGE followed by combined silver and Coomassie blue staining. Antibodies were prepared against the trichocyst fraction and employed to label Lowicryl thin sections of cells. Trichocysts were strongly labeled, as were the surfaces of the plasma and ciliary membranes. Immunoblots of the trichocyst fraction revealed labeling of major bands at 16-29 kD, characteristic of the trichocyst proteins. On immunoblots of the ciliary fraction, approximately eight bands were labeled, including the major cells surface glycoprotein, the immobilization antigen. Ciliary proteins not located on the membrane surface, such as the tubulins, were not labeled. Absorption of the antiserum against fixed P. dubius cells eliminated the cell surface labeling on Lowicryl sections and on immunoblots of the ciliary fraction. The major trichocyst protein bands were as strongly labeled as with the nonabsorbed antiserum. Labeling of several of the minor, higher molecular weight bands of the trichocyst fraction was eliminated, indicating that they are cell surface contaminants. Of the two major structural components of the trichocyst, the shaft and the arms, the antiserum is shown to react nearly exclusively with the shaft proteins on both Lowicryl sections and immunoblots.