Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse.
We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.
Alexandre
A
Zampieri
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.
Julien
J
Champagne
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.
Baptiste
B
Auzemery
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.
Ivanna
I
Fuentes
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.
Benjamin
B
Maurel
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Université de Montpellier, UMR-5203, Montpellier, F-34094, France.|Laboratory of Excellence from genome and epigenome to molecular medicine (EpiGenMed), F-34094 Montpellier, France.
Frédéric
F
Bienvenu
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D019938:Cyclin D1 / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer; D005838:Genotype; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D013737:Testis / Q000378:metabolism / Q000473:pathology
D000906:Antibodies; D000939:Epitopes; D028581:Lanthanoid Series Elements; D019938:Cyclin D1