Detection of free fatty acid receptor 1 expression: the critical role of negative and positive controls.
OBJECTIVE
Adequate evaluation of protein expression is a crucial prerequisite for functional studies. Commonly used strategies comprise detection of proteins by specific antibodies using western blotting and immunohistochemical staining, or detection of mRNA by in situ hybridisation and RT-PCR. We evaluated the tools for the detection of free fatty acid receptor 1 (FFAR1) expression.


METHODS
Commercially available antibody preparations were used to detect endogenous expression of the FFAR1 receptor and this was compared with cell preparations deficient or overexpressing the mouse or human receptor. Concentrations of mRNA were evaluated by RT-PCR.


RESULTS
All insulin-secreting cells, INS-1E, Min6 and mouse islets showed specific expression of Ffar1 at the mRNA level. However, none of the commercially available antibodies specifically detected rat, mouse or human FFAR1.


CONCLUSIONS
Proper positive and negative controls are an important prerequisite for the evaluation of FFAR1 expression.
Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, Department of Internal Medicine IV, University of Tübingen, Otfried-Müller-Str. 10, 72076, Tübingen, Germany.
Charlott-Amelie
CA
Teutsch
Madhura
M
Panse
Manuel
M
Grundmann
Gabriele
G
Kaiser
Evi
E
Kostenis
Hans-Ulrich
HU
Häring
Susanne
S
Ullrich
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C473034:FFAR1 protein, human; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled