Validation of dopamine receptor DRD1 and DRD2 antibodies using receptor deficient mice.
Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria.
Tamara
T
Stojanovic
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
Michaela
M
Orlova
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria.
Fernando J
FJ
Sialana
Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, 2325, Himberg, Austria.
Harald
H
Höger
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
Stanislav
S
Stuchlik
Department of Neurology, Medical University of Vienna, Währinger Gürtel 18, 1090, Vienna, Austria.
Ivan
I
Milenkovic
Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18, 1090, Vienna, Austria.
Jana
J
Aradska
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria. gert.lubec@lubeclab.com.
Gert
G
Lubec
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D000918:Antibody Specificity; D003342:Corpus Striatum / Q000276:immunology*; D051379:Mice; D018345:Mice, Knockout; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000276:immunology*; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; C000600618:DRD2 protein, mouse; C583261:Drd1 protein, mouse; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2
Antibody; Dopamine receptor; Immunohistochemistry; Validation; Western blotting