A general fluorescent light-up probe for staining and quantifying protein

Abstract

Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. The visual analysis of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be realized in 5 min by using PyMDI-Zn as a light-up dye. Based on its cell penetration and low toxicity, PyMDI-Zn could also be applied to locate protein-rich regions and organelles in live cell imaging. Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds.

Keywords: PyMDI-Zn; SDS-PAGE; live cell imaging; protein quantitation.

Figure 1.

Spectral behaviour of PyMDI-Zn (a) Absorption (ab) and emission (em) spectra of PyMDI. (b) E. coli cells stained with PyMDI-Zn were performed on MoFlo^™ XDP Cell Sorter (Beckman Coulter), including E. coli cells without staining (black), E. coli cells stained with PyMDI-Zn, excited at 355 nm and detected at channel FL10 (457/50) (blue) and 488 nm for channel FL1 (529/28) (green). (c) Fluorescent intensity was collected in the presence of excess DNA, RNA, glucose, glycogen, starch and BSA mixed with PyMDI-Zn, PyMDI-Zn for background control. (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-Zn–BSA complex.

Figure 2.

Protein staining in SDS-PAGE with PyMDI-Zn. (a) Nine proteins were separated by 12% SDS-PAGE, RecR (22 kDa); RuvA (24 kDa); RuvB (38 kDa); RecA (39 kDa); T4 DNA ligase (55 kDa); BSA (66 kDa); HSA (66 kDa); MutL (69 kDa); Taq polymerase (94 kDa). (b) Protein samples were commercial protein markers, which contain proteins of 80 kDa (100 ng µl⁻¹), 60 kDa (100 ng µl⁻¹), 40 kDa (200 ng µl⁻¹), 30 kDa (100 ng µl⁻¹) and 20 kDa (100 ng µl⁻¹). Different amounts of protein marker were separated by 12% SDS-PAGE. (c) Total proteins of E. coli (JM109; DH10B and BL21) were separated by 12% SDS-PAGE. The left gel was stained with PyMDI-Zn for 5 min, the right one was stained with CBB for 12 h, then destained overnight.

Figure 3.

HepG2 cells were counterstained with PyMDI-Zn, DAPI and Pyronin Y. (a) PyMDI-Zn, (b) DAPI, (c) the merged image of PyMDI-Zn and DAPI, (d) Pyronin Y, (e) the merged image of PyMDI-Zn and Pyronin Y, (f) the merged image of these three dyes. Nucleus was stained with DAPI, nucleolus (white arrowhead) was stained with Pyronin Y, nucleus and nucleolus could be stained with PyMDI-Zn in the meantime from the merged picture. Scale bars 20 µm.

Figure 4.

Protein quantitation with PyMDI-Zn. (a) Calibration plot for BSA using PyMDI-Zn. The insert reveals the sensitivity of the assay in giving a limit of detection of approximately 0.9 µg ml⁻¹. (b) Detection of contamination protein samples. Melamine or urea was added into the test solution containing protein and PyMDI-Zn respectively. The mixed samples were incubated at room temperature for about 5 min and this assay was tested at wavelength 486 nm for excitation and 520 nm for emission by Thermo Scientific Varioskan Flash.