0000031344 00000 n Oligo Calculator Oligo Submission Form, Product Brochure %PDF-1.3 % (b) Detection of contamination protein samples. JM109, DH10B, BL21(DE3) Chemically Competent Cell, ProteinRuler I and ProteinRuler II, were purchased from TransGen Biotech (Beijing, China). SDS-PAGE was performed on the Invitrogen SureCast system, and the gel images were recorded by GE Healthcare Bio-Sciences AB Typhoon FLA 7000 and Canon Digital camera EOS 600D. DOAJ 2022 default by all rights reserved unless otherwise specified. 0000016252 00000 n J.Z. Different amounts of protein marker were separated by 12% SDS-PAGE. We offer sensitive dyes with very high detection limit to get signals from low expression proteins. 0000005772 00000 n (a) Calibration plot for BSA using PyMDI-Zn. 0000034220 00000 n Absorption spectrometry is based on measuring the ultraviolet absorbance of aromatic amino acids in a protein at different wavelengths [3,4]. 0000129292 00000 n Flow cytometry experiments were performed on a MoFlo XDP Cell Sorter (Beckman Coulter). Different amounts of protein marker were separated by 12% SDS-PAGE. Therefore, mammalian cells (Hela and HepG2) were incubated with PyMDI-Zn and imaged by a confocal microscopy (electronic supplementary material, figure S5B and figure3a). The insert reveals the sensitivity of the assay in giving a limit of detection of approximately 0.9 g ml1. The (Z)-1,2-dimethyl-4-(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (PyMDI) was synthesized according to the synthesis procedure described in [20]. 0000098766 00000 n Images were obtained using the LAS AF software, then subsequently processed with the Adobe Photoshop program. However, disadvantages, such as insolubility in an aqueous phase, aggregation of dyes and instability under ambient conditions, impose restrictions on their practical application [6]. 0000009397 00000 n 2019 Geno Technology Inc., USA. 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. 0000005662 00000 n 0000006022 00000 n Figure 2. We declare we have no competing interests. (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). 0000009043 00000 n Taken together, these results suggest that PyMDI-Zn could be applied to locate protein-rich regions and organelles in live cell imaging. The green fluorescence of PyMDI-Zn might be induced by some components, and some probable biomolecules (DNA, RNA, protein, sugar or polysaccharide) were carefully investigated in vitro. This assay is suitable for the simple and rapid estimation of protein concentration. 0000003001 00000 n 0000017968 00000 n According to excitation/emission spectra (figure1d), we found that PyMDI-ZnBSA complex had an excitation maximum at 486 nm (blue dashed line) and an emission maximum at 520 nm (green solid line). Proteins are essential players in most biological systems and are involved in a variety of complicated biological processes including signal regulation, small molecule transport, immunity and enzyme catalysis. (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-ZnBSA complex. 0000072780 00000 n After 2 or 3 h, fluorescence was monitored at wavelength 530 nm for excitation and 590 nm for emission by Thermo Scientific Varioskan Flash. Although Coomassie dyes are known to bind with a wide range of proteins, R-250 is not usually recommended if you project requires higher sensitivity. 0000018432 00000 n Therefore, to verify this speculation, Pyronin Y, a nucleolus probe, was used to counterstain with PyMDI-Zn (as indicated by arrowhead in figure3d and electronic supplementary material, figure S6B,C), the merged image of PyMDI-Zn and Pyronin Y indicated that those highlighted particles are indeed the nucleolus (figure3e and electronic supplementary material, figure S6D). Therefore, nitrogen-rich compounds, such as melamine and urea, have been added to correct the apparent milk protein content by fraudulent producers. 0000102485 00000 n Apart from this, there were some highlighted particles that appeared in the nucleus (as indicated by the arrowhead in figure3a and electronic supplementary material, figure S5B), which we suspect might be nucleolus because the nucleolus consists of the high concentration of protein and could appear in the nucleus during cell-cycle progression [2528]. Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. 0000006997 00000 n Although both the PyMDI-Zn method and Pierce Kit provided consistent results which were not interfered with by melamine and urea, the protein concentration measured by PyMDI-Zn probe is closer to that of the Kjeldahl method. When we applied PyMDI-Zn to stain Escherichia coli cells, however, very weak fluorescent signals could be detected with a flow cytometry or confocal microscope with 355 nm laser radiation (figure1b and electronic supplementary material, figure S1). 0000008571 00000 n 0000060949 00000 n Webmail. 0000142640 00000 n 0000032680 00000 n Usually, Coomassie blue staining is the first choice for protein detection in SDS-PAGE for most researchers. 0000009704 00000 n Testimonials Unexpectedly, the flow cytometry received a strong fluorescent emission at 520 nm with excitation at 488 nm (figure1b), and cell imaging with a confocal microscope showed similar results (electronic supplementary material, figure S1). 0000024763 00000 n and Z.T. In our experiments, by using PyMDI-Zn as the probe to stain proteins in SDS-PAGE, it only took 5 min to achieve half maximum staining, and the maximum intensity could be obtained in 10 min, which is comparable to the staining result with CBB (figure2b). 0000016644 00000 n If the address matches an existing account you will receive an email with instructions to reset your password. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. A bioorthogonal turn-on fluorescent strategy for the detection of lysine acetyltransferase activity, BODIPY-based ratiometric fluorescent sensor for highly selective detection of glutathione over cysteine and homocysteine, Ultrasensitive fluorescent proteins for imaging neuronal activity, A novel fluorescent probe for the ratiometric recognition of protein based on intramolecular charge transfer, Inhibition of twisting of a green fluorescent protein-like chromophore by metal complexation, Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometry, Improved staining of proteins in polyacrylamide gels including isoelectric-focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250, Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue, Assembly and disassembly of the nucleolus during the cell cycle, Association of Official Analytical Chemists, https://dx.doi.org/10.6084/m9.figshare.c.4614731, http://creativecommons.org/licenses/by/4.0/, doi:10.1146/annurev-physiol-022516-034055, Multiscale imaging approaches for simultaneously mapping distribution of multiple components in infant formula powders, Ethanol from rice byproduct using amylases secreted by Rhizopus microsporus var. Therefore, the cytotoxic potential of PyDMI-Zn was investigated with Alamar Blue Viability assays, showing that PyMDI-Zn would not reduce the cell viability of HepG2 as long as its concentration is less than 200 (electronic supplementary material, figure S7). The responses of PyMDI-Zn to these commonly used reagents were investigated, revealing that the protein detection methods based on PyMDI-Zn probe could tolerate most interfering compounds which could be frequently encountered during the purification and detection of proteins (electronic supplementary material, table S1 and figure S3). The colorimetric determination of protein concentration uses the chromophores that can bind with protein and exhibit a colour change. Our previous data show that E. coli cells could be stained and lighted up with PyMDI-Zn (electronic supplementary material, figure S1), indicating the potential to apply it in cell imaging. 0000033222 00000 n Spectral behaviour of PyMDI-Zn (a) Absorption (ab) and emission (em) spectra of PyMDI. Proteins were loaded in 12% SDS-PAGE. 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. Besides, fluorescent dyes, such as Alexa Fluor, BODIPY and Sypro Ruby, have also been widely used in cell imaging to provide critical insight into the basic nature of cellular function, while they must be modified with a specific probe for a certain protein, such as phalloidin conjugated to Alexa Fluor for F-actin, glibenclamide conjugated to BODIPY for endoplasmic reticulum, ceramide conjugated to BODIPY for Golgi complex and so on [815]. PyMDI-Zn can quickly respond to different proteins with a wide range of molecular weights and resist the interference of most foreign substances. oligosporus. Each visualization technique has its own unique advantages and limitations, and so may only be suitable for certain applications. The Kjeldahl method was performed on a Foss Kjeltec 2200. 0000025296 00000 n Herein, we report an interesting compound, named (Z)-1,2-dimethyl-4-(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (PyMDI), which could bind with proteins specifically in the presence of Zn2+ ions as a light-up probe through emitting strong green fluorescence. 0000007817 00000 n While researchers use several ways to visualize their protein samples, there isnt a single best approach that is ideal for all cases. 0000014016 00000 n All authors gave final approval for publication. The cells were basically green-stained, and the nucleus areas show higher brightness under laser irradiation (figure3a), which is reasonable because the nucleus consists of approximately 90% proteins by dry weight. Fluorescent protein stains are versatile (can be used to stain native, denaturing, 2D, and IEF gels), highly sensitive (can detect nanogram levels of protein), have wide linear dynamic range, and offer low interference. The fluorescent signals of PyMDI-Znprotein complex increased along with the rising of protein concentration (BSA as a model protein, figure4a). 0000005068 00000 n In such cases, G-250 (the colloidal form) is the better choice since it reduces the amount of free dye in the solution which reduces background staining, eliminating the need for additional destaining steps, and enhances the sensitivity of the stain. 0000031944 00000 n Given this information, how do you know which approach to use for your particular project or experiment? In order to verify the reliability of our method in detecting real samples, we compared PyMDI-Zn-based protein quantitation with existing methods, including the Kjeldahl method and Pierce kit (electronic supplementary material, figure S8). Then we selected more potentially interfering compounds including buffering agents, chelating reagents and organic solvents. They also provide minimal protein-to-protein variation so they can be used for quantitative protein comparison. Coomassie dyes work by binding to proteins through their sulfonic acid groups via Van der Waals interactions. Bradford assay is the most widely used colorimetric method for the detection of protein in solution or gel for electrophoresis by using Coomassie Brilliant Blue (CBB) as the protein-binding dye.
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