(Structure and function of the self-incompatibility proteins of Brassica oleracea): Progress report
S-locus specific glycoproteins were purified from S/sub 6/ stigmas following agarose gel isoelectric focusing of stigma extracts and elution from briefly stained gel slices. The purity of the proteins was determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified protein was then used to immunize BALB/c mice at the Cornell Monoclonal Facility. Cells of a mouse whose serum reacted with stigma SLSG were fused to a myeloma cell line, and hybridoma supernatants were screened by Western analysis. Several positive supernatants have been identified. One monoclonal, MAbH8, has been characterized as being specific to a protein epitope of SLSG. This conclusion was arrived at by reacting MabH8 with a series of bacterial ..beta..-galactosidase-SLSG fusion proteins. The specificity of MAbH8 and the applications in which we have used it are described below. For the production of ..beta..-galactosidase-SLSG fusion proteins, the pWR590 family of expression vectors were used. Gene fusions with the complete or truncated versions of the SLSG coding sequence were produced by inserting the appropriate restriction fragments into the polylinker of pWR590 vectors. Following transformation of E. coli cells with these gene fusions, the fusion proteins were purified from bacterial extracts by electroelution from SDS-PAGE gel slices. Immunoblot analysis of these fusion proteins with MAbH8 has demonstrated that the epitope recognized by this monoclonal is a peptide located in the amino-terminal conserved domain of the SLSG molecule. 6 refs., 1 fig.
- Research Organization:
- Cornell Univ., Ithaca, NY (United States)
- DOE Contract Number:
- FG02-88ER13909
- OSTI ID:
- 6327420
- Report Number(s):
- DOE/ER/13909-1; ON: DE89010398
- Resource Relation:
- Other Information: Portions of this document are illegible in microfiche products
- Country of Publication:
- United States
- Language:
- English
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GLYCOPROTEINS
DETECTION
MONOCLONAL ANTIBODIES
PRODUCTION
PROTEINS
BIOASSAY
BACTERIA
ESCHERICHIA COLI
GENETIC MAPPING
MICE
POLLEN
PROGRESS REPORT
PROTEIN STRUCTURE
ANIMALS
ANTIBODIES
DOCUMENT TYPES
GAMETES
GERM CELLS
MAMMALS
MAPPING
MICROORGANISMS
ORGANIC COMPOUNDS
RODENTS
VERTEBRATES
550200* - Biochemistry