[Abstract Title]. - Society for Neuroscience

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Title: Brain-specific cross-reactivity of connexin32 antibodies: problems and solutions
Authors: *S. L. FOWLER 1 , A. C. MCLEAN 2 , S. A. L. BENNETT 2 ;
1 Dept of Biochem, Micro, Immuno, Univ. of Ottawa, Ottawa, ON, Canada; 2 Univ. Ottawa,
Ottawa, ON, Canada
Abstract: Connexins are members of a highly homologous family of transmembrane-spanning
proteins that oligomerize to form gap junction channels. Connexin32 (Cx32) is the most highly
expressed connexin in the liver and is also present in oligodendrocytes and their progenitor cells.
Previous work from our laboratory has implicated Cx32 as an important mediator of neural
progenitor cell specification. However, further study has been complicated by problems with
antigenic cross-reactivity in central nervous system (CNS) tissue when analyses are verified
using the gold-standard control of the Cx32 null-mutant mouse. Here, we demonstrate that the
vast majority of commercially available antibodies detect a protein in both wild-type (WT) and
Cx32 null-mutant mice that migrates with a mobility of 27 kD. We show that this cross-reactive
epitope is specific to CNS. Liver lysates derived from Cx32 null-mutant mice do not exhibit nonspecific
labeling. We further demonstrate that WT Cx32 in liver lysates migrates with a
molecular weight that is 4 kDa higher than the anti-Cx32-reactive protein in brain and spinal
cord. We find that this artifactual labeling is only observed under denaturing conditions. Crossreactivity
with a reactive protein of approximately the same mobility as WT Cx32 is not detected
in brain lysates of Cx32 null-mutant mice when tertiary structure is maintained and protein
expression is analyzed by immunoprecipitation, immunofluourescence, or ELISA. Finally, by
sucrose gradient fractionation and mass spectrometry analysis of whole brain and corpus
callosum, we have identified the subcellular compartments that contain the cross-reactive protein
and characterized the cross-reactive epitopes. These carefully controlled antibody
characterizations, comparing reactivity in WT and Cx32-null mutant CNS within the context of
multiple protein analysis techniques, permit the proper detection and quantification of Cx32 from
brain and spinal cord, and provide essential information for accurate Cx32 investigation in the
brain.
Disclosures: S.L. Fowler, None; A.C. McLean, None; S.A.L. Bennett, None.
Poster
241. Glial Neuronal Interactions
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 241.10/G4