FIGURE SUMMARY
Title

Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr)

Authors
Hossain, M.F., Hossain, S., Sarwar Jyoti, M.M., Omori, Y., Ahamed, S., Tokumoto, T.
Source
Full text @ Biochem Biophys Rep
Fig. 1

Expression and purification of the ligand-binding domain (LBD) of zebrafish Pgr as a fusion protein with GST and production of GQD-coupled GST-zPgrLBD.

A. A diagram of the produced recombinant GST-fusion zPgrLBD. The predicted ligand-binding domain at the C-terminus is indicated by black boxes. The GST tag at the N-terminus is indicated by a white box. B. SDS-PAGE analysis of purified recombinant GST-zPgrLBD. Protein bands were detected by CBBR staining (CBBR) or immunostained with anti-GST-tag antibody (α-GST). C. Western blot analysis of GST-zPgrLBD and GQD-GST-zPgrLBD. The protein band of GST-zPgrLBD is indicated by an arrowhead. The bands of GQD-GST-zPgrLBD are indicated by a parenthesis.
Fig. 2

Fluorescence characteristics of GQD-GST-zPgrLBD and competition of binding of P4-BSA-FITC with GQD-GST-zPgrLBD by steroids and their analogues.

(A) The fluorescent scanning pattern of free GQD-GST-zPgrLBD is indicated in blue line. Fluorescent scanning pattern of the reaction mixture with (orange line) or without free P4 (green line). The difference of fluorescence at 520 nm caused by the addition of free P4 is indicated by the double-sided arrow. (B) The dose-dependent effects of steroids (progesterone (P4), 17β-estradiol (E2), testosterone (T), cortisol) and their analogues (17,20β-DHP and Org OD 02) were determined using the established assay. An assay was performed in triplicate for each compound. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3

Competition by steroids and progestins for binding to GQD-GST-zPgrLBD.

Samples were incubated with 2 nM [3H]-17,20β-DHP and 1 μM of competitor. An assay was performed in triplicate for each competitor. The average of the three assays is indicated with standard deviation. Competition for [3H]-17,20β-DHP binding are expressed as a percentage of maximum specific progesterone (P4) binding. The competitors analyzed were progesterone (P4); 17,20β-DHP; Org OD 02; 17β-estradiol (E2); testosterone (T); Cortisol.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Biochem Biophys Rep
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