Multicolor Imaging of Bifacial Activities of Estrogens

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1461; p. 153
• Main Authors: Kim, Sung-Bae, Umezawa, Yoshio
• Format: Journal Article
• Language: English
• Published: United States 2016
• Subjects: Animals; Cell Line; Estrogens - genetics; Estrogens - metabolism; Gene Expression; Gene Order; Genetic Vectors - genetics; Humans; Ligands; Luminescent Measurements - methods; Molecular Imaging - methods; Molecular Probes - chemistry; Molecular Probes - genetics; Protein Binding; Protein Interaction Domains and Motifs; Receptors, Estrogen - chemistry; Receptors, Estrogen - genetics; Receptors, Estrogen - metabolism; Estrogen receptor; Multicolor imaging; Bioluminescence; Click beetle; Luciferase; Single-chain probe
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-3813-1_12

The present protocol introduces multicolor imaging of bifacial activities of an estrogen. For the multicolor imaging, the authors fabricated two single-chain probes emitting green or red bioluminescence (named Simer-G and -R, respectively) from click beetle luciferase (CBLuc) green and red: Simer-R consists of the ligand binding domain of estrogen receptor (ER LBD) and the Src homology-2 (SH2) domain of Src, which are sandwiched between split-CBLuc red (CBLuc-R). On the other hand, Simer-G emitting red light consists of the ER LBD and a common consensus sequence of coactivators (LXXLL motif), which are inserted between split-CBLuc green (CBLuc-G). This probe set creates fingerprinting spectra from the characteristic green and red bioluminescence in response to agonistic and antagonistic activities of a ligand of interest. The present protocol further provides a unique methodology to calculate characteristic estrogenicity scores of various ligands from the spectra.