Abstract
Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of 125I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.
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Abbreviations
- D:
-
diffusion coefficient
- Mr :
-
relative molecular mass
- PBS:
-
phosphate-buffered saline (8.00 g NaCl, 1.15 g Na2HPO4, 0.20 g NaH2PO4 per 1 L, pH 7.2)
- TPBS:
-
phosphate-buffered saline containing 0.5% Tween-20
- TX-100, TX-114:
-
Triton X-100, X-114
- SDS:
-
sodium dodecyl sulfate
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Villanueva, M.A., Metcalf, T.N. & Wang, J.L. Monoclonal antibodies directed against protoplasts of soybean cells. Planta 168, 503–511 (1986). https://doi.org/10.1007/BF00392269
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DOI: https://doi.org/10.1007/BF00392269