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5A3 INVESTIGATOR Name John E. Wilson Address 301 Biochemistry, Dept. of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 E-Mail: [email protected] IMMUNOGEN Substance Name Origin Type I isozyme of rat hexokinase Purified from rat brain. Purification protocol given in Wilson, JE (1989) Preparative Biochemistry 19: 13-21. Chemical Composition Developmental Stage IMMUNIZATION PROTOCOL Details of the immunization and subsequent fusion protocol are found in Finney et al. (1984) J. Biol. Chem. 259: 8232-8237. Donor Animal Species mouse Strain Swiss white Sex either sex Organ and tissue splenic lymphocytes Immunization Dates immunized Amount of antigen 100 μg purified rat Type I hexokinase Route of immunization injected intraperitoneally Adjuvant Freund’s complete adjuvant; booster immunizations were the same except using Freund’s incomplete adjuvant. FUSION Date Myeloma cell line Species Designation MONOCLONAL ANTIBODY Isotype Specificity Cell binding Immunohistology Antibody competition Species Specificity ANTIGEN Chemical properties mouse Sp2/O-Ag14 IgG1 Monoclonal 5A3 recognizes an epitope in the N-terminal half of rat Type I hexokinase. Crossreactivity with the Type II or Type III isozymes is thought to be unlikely. rat; crossreactivity with Type I isozyme from other mammalian species has not been investigated N-terminal half of the Type I isozyme of hexokinase from rat The epitope recognized is a “conformational” (or “discontinuous”) epitope, and immunoreactivity is markedly affected by conformational changes in the Type I hexokinase, e.g., as a result of binding certain ligands [Smith and Wilson (1992) Arch. Biochem. Biophys. 292: 165-178]. Molecular weight Characterization Immunoprecipitation Immunoblotting The antibody is reactive with the native enzyme (ELISA) but not the denatured form of the enzyme and thus is not useful for immunoblots. Purification Amino acid sequence analysis Functional effects Immunohistochemistry PUBLICATIONS : (Continued) 5A3 (Continued) Finney, K.G., Messer, J.L., DeWitt, D.L., and Wilson, J.E. (1984). Monoclonal antibodies against rat brain hexokinase: effects on catalytic function and binding to the outer mitochondrial membrane. J. Biol. Chem. 259, 8232-8237. Wilson, J.E., and Smith, A.D. (1985). Monoclonal antibodies against rat brain hexokinase: utilization in epitope mapping studies and establishment of structure-function relationships. J. Biol. Chem. 260, 12838-12843. Ureta, T., Smith, A.D., and Wilson, J.E. (1986). Hexokinase A from mammalian brain: comparative peptide mapping and immunological studies with monoclonal antibodies. Arch. Biochem. Biophys. 246, 419-427. Smith, A.D., and Wilson, J.E. (1986). A modified ELISA that selectively detects monclonal antibodies recognizing native antigen. J. Immunol. Meth. 94, 31-35. Smith, A.D., and Wilson, J.E. (1991). Disposition of mitochondrially bound hexokinase at the membrane surface, deduced from reactivity with monoclonal antibodies recognizing epitopes of defined location. Arch. Biochem. Biophys. 287, 359-366. Smith, A.D., and Wilson, J.E. (1992). Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and regulatory function. Arch. Biochem. Biophys. 292, 165-178. Hashimoto, M., and Wilson, J.E. (2000). Membrane potential-dependent conformational changes in mitochondrially bound hexokinase of brain. Arch. Biochem. Biophys. 384, 163-173. ACKNOWLEDGMENTS STATEMENT We have been asked by NICHD to ensure that all investigators include an acknowledgment in publications that benefit from the use of the DSHB's products. We suggest that the following statement be used: “The (select: hybridoma, monoclonal antibody, or protein capture reagent,) developed by [Investigator(s) or Institution] was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.” Please send copies of all publications resulting from the use of Bank products to: Developmental Studies Hybridoma Bank Department of Biology The University of Iowa 028 Biology Building East Iowa City, IA 52242