Abstract
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.
Highlights
Decided to test this alternative that we named Tandem-HTRF, for the study of Cyclin D1 in vivo
After testing several protein extraction methods to obtain HTRF-compatible lysates directly from mice tissues, we found that Flag-HA Tandem-HTRF led to a satisfying signal to noise ratio for the detection of Tagged-Cyclin D1 protein (CycD1) (Fig. 1b,c, Supplementary Fig. 2a,b)
Using Tandem-HTRF as a semi-quantitative method, we found that Tagged-CycD1 half-life was relatively short, as reported for wildtype CycD1 after translation inhibition by cycloheximide (Supplementary Fig. 3a–c)[8]
Summary
Decided to test this alternative that we named Tandem-HTRF, for the study of Cyclin D1 in vivo. CycD1 has been shown as an oncogene to participate in tumor growth by direct activation of its major partner, the Cyclin-Dependent Kinase 4 (CDK4)[6,12,13] For these reasons, the CycD1/CDK4 complex represents a therapeutic target of interest in cancer biology[14]. Using current biochemistry techniques, a specific signal for CycD1 protein is hardly detectable in adult post-mitotic mouse organs compared to developing tissues. In most cases this pattern of expression in adult animals is arguably a faithful reflection of the overall status of the few cycling progenitor cells remaining[15,16,17]. We found that CycD1 remains expressed ubiquitously in adult tissue and appears unexpectedly to be down-regulated upon environmental challenge
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