Download 5A3 INVESTIGATOR Name John E. Wilson Address 301

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