Detection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopes.
GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT(1) and MT(2) receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein.
Hamouda
Hassina Ould
HO
Institut Cochin, Department of Cell Biology, Paris, France.
Chen
Patty
P
Levoye
Angélique
A
Sözer-Topçular
Nejla
N
Daulat
Avais M
AM
Guillaume
Jean-Luc
JL
Ravid
Rivka
R
Savaskan
Egemen
E
Ferry
Gilles
G
Boutin
Jean A
JA
Delagrange
Philippe
P
Jockers
Ralf
R
Maurice
Pascal
P
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism*; D001666:Binding Sites, Antibody; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D020539:Sequence Analysis, Protein
D000906:Antibodies; D000939:Epitopes; C509754:GPR50 protein, human; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled