0000007910 00000 n Figure 2. Protein Cross-Linking & Protein Modification, Ion Exchange Chromatography Resins and Methods, Protein Extraction & Lysis Buffer (PE LB) Systems, Molecular Biology Accessories, Buffers & Reagents, Biotechnology, Science for the New Millennium, Biotechnology Basics by Ellyn Daugherty, Purification Resin Synthesis & Production. 0000085625 00000 n Therefore, mammalian cells (Hela and HepG2) were incubated with PyMDI-Zn and imaged by a confocal microscopy (electronic supplementary material, figure S5B and figure3a). Then we selected more potentially interfering compounds including buffering agents, chelating reagents and organic solvents. 2017M612998). (b) Detection of contamination protein samples. 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). conceived of the study, designed the study, coordinated the study and helped draft the manuscript. 0000006472 00000 n Learn more about DOAJs privacy policy. However, disadvantages, such as insolubility in an aqueous phase, aggregation of dyes and instability under ambient conditions, impose restrictions on their practical application [6]. Protein staining in SDS-PAGE with PyMDI-Zn. 0000006997 00000 n Given this information, how do you know which approach to use for your particular project or experiment? 2019 Geno Technology Inc., USA. Coomassie stain protocol: the SDS-PAGE gel was rinsed with deionized water, and then stained with Coomassie solution, incubation with gentle agitation at room temperature for 120 h. Gels were destained overnight with gentle agitation. Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.4614731. 0000014447 00000 n 0000006340 00000 n 0000034291 00000 n 0000008005 00000 n http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Methods currently used for the determination of the protein content of foodstuffs, including the Kjeldahl and Dumas Methods, depend on the determination of nitrogen [29,30]. 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. These results demonstrate that the fluorophore PyMDI-Zn described here is a versatile light-up probe for protein research and food quality control. Silver staining protocols (i.e., silver nitrate and silver-ammonia complex methods) involve the use of simple equipment and reagents, have high sensitivity about 10 to 100 times more sensitive than Coomassie dyes and are compatible with downstream applications such as mass spectrometry. Content on this site is licensed under a Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license. 0000008382 00000 n 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. GCC Team The insert reveals the sensitivity of the assay in giving a limit of detection of approximately 0.9 g ml1. The colorimetric determination of protein concentration uses the chromophores that can bind with protein and exhibit a colour change. Taken together, these results suggest that PyMDI-Zn could be applied to locate protein-rich regions and organelles in live cell imaging. 0000008477 00000 n All proteins in SDS-PAGE could be successfully stained with 100 M PyMDI-Zn, and the bands corresponding to different proteins could be seen clearly under UV irradiation with or without washing step (electronic supplementary material, figure S2), which is comparable to the staining result with CBB. 0000017368 00000 n 0000014519 00000 n 0000008571 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. Copyrights and related rights for article metadata waived via CC0 1.0 Universal (CC0) Public Domain Dedication. While silver ions cannot stain glycoproteins, phosphoproteins, and other hard-to-stain proteins, zinc ions can fill this role quite nicely. Each visualization technique has its own unique advantages and limitations, and so may only be suitable for certain applications. PyMDI-Zn can quickly respond to different proteins with a wide range of molecular weights and resist the interference of most foreign substances. Moreover, the total proteins of E. coli bacteria were separated by 12% SDS-PAGE and stained with 100 M PyMDI-Zn for 5 min. Sensitivity: Linear responses over the range of 0.5g-50g protein, Flexible Protocols: Suitable for tube or Titer plate assays, Ready to use assay reagents and no preparation required. 0000085601 00000 n (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-ZnBSA complex.Download figureOpen in new tabDownload PowerPoint. All Rights Reserved. 0000003736 00000 n 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). The left gel was stained with PyMDI-Zn for 5 min, the right one was stained with CBB for 12 h, then destained overnight.Download figureOpen in new tabDownload PowerPoint. Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. The concentration of protein was plotted against the corresponding fluorescent intensity to obtain a standard curve. Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the number of proteins analyzed, the accuracy of spot quantification and reproducibility. Protein quantitation with PyMDI-Zn. As shown in figure4b, the addition of melamine or urea did not cause the increase of the fluorescent response of PyMDI-Zn to BSA (a standard protein). Training Researchers prefer Coomassie dyes since they are simple, economical, and very easy to use. This assay is suitable for the simple and rapid estimation of protein concentration. Melamine or urea was added into the test solution containing protein and PyMDI-Zn respectively. Fluorescent spectrometry is of particular interest for the staining and visualizing of proteins because of its high sensitivity and convenience. (b) Protein samples were commercial protein markers, which contain proteins of 80 kDa (100 ng l1), 60 kDa (100 ng l1), 40 kDa (200 ng l1), 30 kDa (100 ng l1) and 20 kDa (100 ng l1). 0000025541 00000 n For one, it does not react uniformly with all proteins and cannot detect certain proteins (e.g., glycoproteins) due to its limited dynamic range. 0000033684 00000 n 0000004951 00000 n News and Events The insert reveals the sensitivity of the assay in giving a limit of detection of approximately 0.9 g ml1. 0000005185 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). 0000014268 00000 n Spectral behaviour of PyMDI-Zn (a) Absorption (ab) and emission (em) spectra of PyMDI. 0000129292 00000 n Therefore, nitrogen-rich compounds, such as melamine and urea, have been added to correct the apparent milk protein content by fraudulent producers. Figure 3. It also requires the use of hazardous chemicals and is temperature-dependent. Protein analysis with rapid response, high sensitivity and easy handling is of fundamental importance for understanding the diverse functions of proteins. 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. And since they produce intensely colored complexes upon binding to protein molecules, they can also be easily detected in just a few minutes; maximal staining can be achieved within an hour. 0000010940 00000 n 0000005068 00000 n 0000002729 00000 n 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. 0000099742 00000 n 0000004030 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]. Stay up to date with G-Biosciences by signing up for our newsletter. HepG2 was cultured in a 96-well plate, the cells were exposed to PyMDI-Zn at a concentration in the range of 50 500 (DMSO as a control) for 24 h, then the medium was replaced with 200 l DMEM containing 10% (v/v) Alamar Blue. The left gel was stained with PyMDI-Zn for 5 min, the right one was stained with CBB for 12 h, then destained overnight. (a) Calibration plot for BSA using PyMDI-Zn. To test the application of PyMDI-Zn in protein analysis, we first applied PyMDI-Zn for protein staining in SDS-PAGE. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. trailer << /Size 183 /Info 61 0 R /Root 65 0 R /Prev 497924 /ID[<7e6e70feff513a00a2beed98f2a4a3f8><7b7842c5a3b99e058cce5a899fec3c77>] >> startxref 0 %%EOF 65 0 obj << /Type /Catalog /Pages 63 0 R /Metadata 62 0 R /Outlines 69 0 R /Threads 66 0 R /OpenAction [ 68 0 R /FitH 804 ] /PageMode /UseOutlines /PageLabels 60 0 R >> endobj 66 0 obj [ 67 0 R ] endobj 67 0 obj << /I << /Title (tx1)>> /F 90 0 R >> endobj 181 0 obj << /S 447 /T 699 /O 751 /L 767 /Filter /FlateDecode /Length 182 0 R >> stream They also provide minimal protein-to-protein variation so they can be used for quantitative protein comparison. 0000099628 00000 n 0000114012 00000 n 0000032680 00000 n The green fluorescent protein-like chromophore (PyMDI) has been developed and carefully investigated by the Tolbert group. Hb```f` b,wXO68eiCbFT3(yj. If the address matches an existing account you will receive an email with instructions to reset your password. 0000004816 00000 n Thus, based on our data, we believe that PyMDI-Zn could be applicable as a good fluorescent gel stain. Oligo Calculator Together, PyMDI-Zn could light up (520 nm) when binding to proteins and excited around 486 nm, indicating that PyMDI-Zn might be a promising tool for protein detection and analysis. 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. Scale bars 20 m.Download figureOpen in new tabDownload PowerPoint. (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-ZnBSA complex. 0000025885 00000 n J.Z., G.C., F.D., Y.Y. In this review article, the efficiency of the most widely used dyes was investigated. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. In general, foreign substances, such as detergents, chelating reagents, inorganic salts and organic solvents, could significantly change the response of dye to proteins, thus it usually takes a long time to get rid of them for proteins staining in SDS-PAGE. 0000008099 00000 n Therefore, to achieve high efficiency for protein detection in vitro or in vivo, novel fluorescent probes with high specificity, resistance to interference from foreign substances, ease of use and broad dynamic range are highly desired [1619]. Furthermore, Coomassie dyes offer a medium to relatively high sensitivity, as they can detect proteins as low as 1 g, and are compatible with most downstream applications (e.g., mass spectrometry, qualitative visualization, quantitative densitometry) since they do not alter the chemical structure of the target protein. By using PyMDI-Zn as a dye, the fast protein staining in SDS-PAGE could be realized in 5 min, providing great convenience. 0000005896 00000 n 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. Laser 355 and 488 nm were selected when using a flow cytometer, and laser 405, 488 and 552 nm for Confocal Microscope. While researchers use several ways to visualize their protein samples, there isnt a single best approach that is ideal for all cases. 0000007363 00000 n 2017TD0021); Chengdu Municipal Bureau of Science and Technology (grant nos. 0000009963 00000 n 0000010413 00000 n 0000014016 00000 n 0000017968 00000 n 0000015767 00000 n However, this assay exhibits various responses for different proteins and most commercial detergents could interfere with the protein assay [5]. 0000034510 00000 n 0000004288 00000 n 0000006198 00000 n 0000048199 00000 n Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds. 0000033440 00000 n However, the washing step is no longer needed when using PyMDI-Zn to stain protein in our experiment, implying that this new probe may be more tolerant of interference from other non-protein substances. 0000018432 00000 n 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). 0000009043 00000 n And the response of some fluorescent reagents, such as Sypro Ruby, against proteins, will be greatly affected by detergents like sodium dodecyl sulfate (SDS), thus it takes a long time to get rid of SDS for staining proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [7]. Contact Us Staining and visualization of PAGE gel is essential for quantification and data extraction from the gel. 0000060949 00000 n (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. 0000008287 00000 n Different protein-targeting detection methods, including the fast and simple fluorescent staining in SDS-PAGE, live cell imaging and accurate quantitation in solution, have been realized based on this new fluorescent molecular probe. 0000060973 00000 n think G-Biosciences! 0000016252 00000 n 0000098742 00000 n We found that the fluorescence intensity of PyMDI-ZnBSA is very stable in most detergents and retains 90% fluorescence at the concentration of 10% for SDS, 10% for Triton X-100, 2.5% for NP-40, 0.25% for Tween 20, respectively. Proteins were loaded in 12% SDS-PAGE. (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. Figure 1. As illustrated in figure2a, nine proteins of different sizes (2294 kDa) were stained with PyMDI-Zn. 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.Download figureOpen in new tabDownload PowerPoint. 0000004515 00000 n Protein solutions are normally assayed in triplicate. Dyes, Ions, or Fluorescent Stains: What Are the Best Ways to Visualize Protein In Gels? 0000009397 00000 n 0000003714 00000 n 0000142640 00000 n (b) Protein samples were commercial protein markers, which contain proteins of 80 kDa (100 ng l1), 60 kDa (100 ng l1), 40 kDa (200 ng l1), 30 kDa (100 ng l1) and 20 kDa (100 ng l1). 0000126511 00000 n 0000006022 00000 n 0000032118 00000 n 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). 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. (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). Protein staining in SDS-PAGE with PyMDI-Zn. 0000015395 00000 n Images were obtained using the LAS AF software, then subsequently processed with the Adobe Photoshop program. Figure 4. However, these fluorescent dyes designed for fluorescence imaging of tissue distribution and subcellular localization have some significant disadvantages which include (i) requiring of cell fixation, (ii) more or less cytotoxicity, (iii) high cost due to complex chemical synthesis. 0000009704 00000 n Different amounts of protein marker were separated by 12% SDS-PAGE. 0000005326 00000 n DOAJ 2022 default by all rights reserved unless otherwise specified. 0000009136 00000 n This work was supported by the National Natural Science Foundation of China (grant nos. Bovine serum albumin (BSA), human serum albumin (HSA), Brilliant blue R250, Pyronin Y, DAPI and SYPRO (R) Orange Protein Gel Stain were purchased from Sigma Chemicals Co. All other chemicals are of analytical reagent grade. 0000007817 00000 n One hundred microlitres of solutions containing different amounts of protein (0600 g) with 100 M PyMDI-Zn were incubated at room temperature for about 5 min. 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. 0000005500 00000 n 0000098766 00000 n Silver and zinc ions are typically used for detecting proteins separated by SDS-PAGE. 0000006906 00000 n Webmail. The datasets supporting this article have been uploaded as part of the electronic supplementary material. Despite its desirable qualities, silver staining has its limitations. 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). (b) Detection of contamination protein samples. To address these limitations, G-Biosciences offers a glutaraldehyde-free silver staining kit that provides maximum sensitivity and visibility. As illustrated in figure1c, the fluorescent intensity of PyMDI-Zn increased about six times at 520 nm with excitation at 488 nm in the presence of BSA, while there was no increased signal that could be detected using other samples (figure1c). Different amounts of protein marker were separated by 12% SDS-PAGE. The present study demonstrates a light-up fluorophore PyMDI-Zn which could specifically bind to proteins and provide a red-shifted fluorescent emission. 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]. 0000072780 00000 n (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). 0000008854 00000 n As expected, the Kjeldahl method was easily interfered with by nitrogen-rich compounds such as melamine and urea. 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). Then, the fluorescence of those samples was detected at 520 nm with excitation at 486 nm by Thermo Scientific Varioskan Flash. 0000007725 00000 n carried out the molecular and cell laboratory works, participated in data analysis, carried out the design of the study and drafted the manuscript; J.Z., K.X., X.H., J.D., X.C. 0000005662 00000 n The protein concentration of the unknown samples was determined by the standard curve. 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. Spectral behaviour of PyMDI-Zn (a) Absorption (ab) and emission (em) spectra of PyMDI. Figure 4. Cell cultures were maintained in the DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin storage solution (100 U/ml penicillin, 100 mg/ml streptomycin), at 37C in a humidified atmosphere containing 5% CO2. 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]. oligosporus. 0000007272 00000 n Melamine or urea was added into the test solution containing protein and PyMDI-Zn respectively. Oligo Submission Form, Product Brochure 0000005772 00000 n Figure 3. [1,2]. HepG2 cells were counterstained with PyMDI-Zn, DAPI and Pyronin Y. 0000008949 00000 n Protein laboratories around the world have traditionally used Coomassie dyes (i.e., R-250 and colloidal G-250) for visualizing protein bands resolved by SDS-PAGE.