Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!
The study of endogenous cannabinoid CB1 receptor proteins in neuronal tissues and cells relies on the availability of highly specific antibodies. We have tested the ability of a series of CB1 antibodies to detect endogenous receptors in brain as well as hemagglutinin (HA)-tagged receptors transfected into HEK-293 cells using a combination of immunological methods. An initial comparison of several CB1 antibodies in mouse brain revealed substantial differences in staining pattern to ligand binding by autoradiography. Antibodies were then tested immunocytochemically against HEK cells expressing HA-tagged rat and human CB1 receptors. None of the commercial antibodies tested were able to detect the receptor in this context. All antibodies were then screened by Western blotting using lysates from the HEK cells and rodent brain homogenates. Again, none of the commercially available antibodies detected proteins of the correct molecular weight in transfected cell lines or brain homogenates, although all recognized multiple proteins in brain tissues. We conclude that the commercially available antibodies we tested failed to detect CB1 receptors abundantly expressed in HEK cells or native receptors in brain slices or homogenates. As such, comprehensive validation of the specificity of these CB1 antibodies for a particular application is essential before use.
Grimsey
Natasha L
NL
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Goodfellow
Catherine E
CE
Scotter
Emma L
EL
Dowie
Megan J
MJ
Glass
Michelle
M
Graham
E Scott
ES
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D001345:Autoradiography / Q000379:methods; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002461:Cell Line, Transformed; D003511:Cyclohexanols / Q000493:pharmacokinetics; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006384:Hemagglutination; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
D000906:Antibodies; D003511:Cyclohexanols; D043884:Receptor, Cannabinoid, CB1; D014316:Tritium; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol