Tissue-specific cross-reactivity of connexin32 antibodies: problems and solutions unique to the central nervous system.
Gap junction proteins are a highly homologous family of 21 connexins. Here, the authors describe a tissue-specific technical artifact complicating analysis of connexin32 protein expression in the central nervous system. The authors show that in brain, but not liver, eight commonly employed antibodies exhibit a higher affinity for a cross-reactive protein that masks the detection of connexin32. Cross-reactivity is evident in Western blot analyses when proteins are subjected to reducing/denaturing conditions but not immunoprecipitation or immunofluorescent applications. Through bioinformatic analyses, tested by sucrose gradient fractionation and immunoblotting of lysates from connexin null-mutant mice, the authors show that the cross-reactive protein is not found in the same cellular compartments as connexin32 and is likely not a member of the connexin family. These findings are presented with the intent of helping to reduce the amount of time laboratories currently expend in validating changes in connexin32 expression in the central nervous system.
Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
Stephanie L
SL
Fowler
Ashleigh C
AC
McLean
Steffany A L
SA
Bennett
D000368:Aged; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D016477:Artifacts; D001923:Brain Chemistry; D002490:Central Nervous System / Q000378:metabolism*; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000939:Epitopes; D005260:Female; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D011487:Protein Conformation; D000097002:Gap Junction beta-1 Protein
D000906:Antibodies; D017630:Connexins; D000939:Epitopes; D000097002:Gap Junction beta-1 Protein