Immunocytochemical localization of the beta 2 subunit of the gamma-aminobutyric acidA receptor in the rat brain.
An antiserum to the beta 2 subunit of the rat gamma-aminobutyric acid (GABAA) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the beta 2 subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABAA receptors. The anti-beta 2 antibodies were affinity purified on immobilized beta 2 intracellular loop peptide. The antibodies recognized a 55-57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABAA receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the beta 2 subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-beta 2 antibody and the monoclonal antibody 62-3G1 (which recognizes both beta 2 and beta 3 subunits) is presented. The procedure described for generating and preparing specific anti-beta 2 subunit antibodies that are valuable for immunocytochemistry could be extended to other GABAA receptor subunits.
Moreno
J I
JI
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110-2499.
Piva
M A
MA
Miralles
C P
CP
De Blas
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001923:Brain Chemistry / Q000502:physiology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D011233:Precipitin Tests; D051381:Rats; D011963:Receptors, GABA-A / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013205:Staphylococcal Protein A
D010446:Peptide Fragments; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A