Media Coverage, GCC Catalogue 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]. 0000126614 00000 n
0000033440 00000 n
0000015079 00000 n
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]. 0000034291 00000 n
The change in color density is proportional to protein concentration. Then, the fluorescence of those samples was detected at 520 nm with excitation at 486 nm by Thermo Scientific Varioskan Flash. Webmail. 0000008287 00000 n
0000008382 00000 n
0000014016 00000 n
0000005662 00000 n
and Z.T. think G-Biosciences! 0000016252 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). 0000034220 00000 n
0000017300 00000 n
Super Stain Nucleic Acid Gel Stain, 20000X, About Us 0000005772 00000 n
The concentration of protein was plotted against the corresponding fluorescent intensity to obtain a standard curve. 0000025541 00000 n
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. (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-ZnBSA complex. 0000004288 00000 n
0000007634 00000 n
RecR, RuvA, RuvB, RecA and MutL were cloned into an expression vector, and the proteins were purified. Different amounts of protein marker were separated by 12% SDS-PAGE. (d) Excitation spectrum (blue dashed line) and emission spectrum (green solid line) of PyMDI-ZnBSA complex.Download figureOpen in new tabDownload PowerPoint. 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]. 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. 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. Proteins were loaded in 12% SDS-PAGE. We declare we have no competing interests. Sypro Ruby, Cyanines, BODIPY, pyridinium bromide and Fluorescamine. 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. 0000014447 00000 n
0000114036 00000 n
Copyrights and related rights for article metadata waived via CC0 1.0 Universal (CC0) Public Domain Dedication. An improved Coomassie Dye based protein assay based on the Bradford Protein Assay. 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. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes (Sypro Ruby, Deep Purple) and cyanine labeled methods were compared. JM109, DH10B, BL21(DE3) Chemically Competent Cell, ProteinRuler I and ProteinRuler II, were purchased from TransGen Biotech (Beijing, China). 0000099108 00000 n
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. Laser 355 and 488 nm were selected when using a flow cytometer, and laser 405, 488 and 552 nm for Confocal Microscope. 0000008854 00000 n
0000015767 00000 n
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). DOAJ 2022 default by all rights reserved unless otherwise specified. 0000014519 00000 n
For quotation requests and questions, you may contact us by support@gccbiotech.co.in. 0000098766 00000 n
0000009136 00000 n
Staining and visualization of PAGE gel is essential for quantification and data extraction from the gel. Aside from the two additional methyl groups in the G-250, these two variants have almost identical chemical structures. 0000033399 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]. Therefore, mammalian cells (Hela and HepG2) were incubated with PyMDI-Zn and imaged by a confocal microscopy (electronic supplementary material, figure S5B and figure3a). Researchers prefer Coomassie dyes since they are simple, economical, and very easy to use. 0000002729 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. 0000003736 00000 n
21572222, 21708037, 21877108); the Innovative Team of Sichuan Province (grant no. 0000033684 00000 n
0000015395 00000 n
Coomassie dyes work by binding to proteins through their sulfonic acid groups via Van der Waals interactions. 0000003001 00000 n
This website uses cookies to ensure you get the best experience. To address these limitations, G-Biosciences offers a glutaraldehyde-free silver staining kit that provides maximum sensitivity and visibility. In choosing the appropriate fluorescent dye for your particular application, you need to consider its compatibility with (1) the experimental design, (2) available illumination source, and (3) other fluorescent materials in the sample which may produce background autofluorescence. 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. 0000017604 00000 n
0000129292 00000 n
0000007454 00000 n
0000033222 00000 n
If the address matches an existing account you will receive an email with instructions to reset your password. All authors gave final approval for publication. The SDS prevents the stain from binding to the proteins, producing clear protein bands on the semi-opaque gel within 10 to 15 minutes. 0000007910 00000 n
The plot indicates a good linear relationship between the emission intensities and the BSA concentration (up to 1 mg ml1 of BSA, r > 0.994). SARPStain Protein Dye, 100 small gel equivalent, SARPStain Protein Dye, 250 small gel equivalent, SARPStain Plus Protein gel staining kit (100 small gel), G-SNAP in-gel protein visualization reagent (100 small gel), G-SNAP in-gel protein visualization reagent (200 small gel), Ponceau S, ready to use protein on membrane staining solution, Coommassie protein gel staining solution, Ready to use. Moreover, the total proteins of E. coli bacteria were separated by 12% SDS-PAGE and stained with 100 M PyMDI-Zn for 5 min. This assay is suitable for the simple and rapid estimation of protein concentration. Hb```f` b,wXO68eiCbFT3(yj. Silver ions in traditional silver staining kits may also fail to facilitate complete proteolytic digestion and maximal peptide recovery, which is a must for optimal mass spectrometry analysis. Different amounts of protein marker were separated by 12% SDS-PAGE. Protein estimation can be performed using as little as 0.5g protein. 0000006737 00000 n
Protein quantitation with PyMDI-Zn. 0000126535 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. (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. The present study demonstrates a light-up fluorophore PyMDI-Zn which could specifically bind to proteins and provide a red-shifted fluorescent emission. 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. 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. 0000004030 00000 n
0000002915 00000 n
In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. Enter your email address below and we will send you the reset instructions. (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. (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. Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.4614731. (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).