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and transmitted securely. In addition, the user-dependent nature of the process can introduce variability, when comparing results generated by different users. 45 37
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Gel images of complex protein samples imaged by UV activation or Coomassie G-250 staining: Lane 1, Precision Plus Protein unstained standards (Bio-Rad Laboratories); Lane 2, mouse liver extract (12.5 g); Lane 3, rat liver (12.5 g); Lane 4, mouse thymus (12.5 g); Lane 5, rat thymus (12.5 g); Lane 6, HeLa cell lysates (12.5 g); Lane 7 E. coli lysate (67.5 l); Lane 8, ProteoMiner kit-treated human serum (Bio-Rad Laboratories), 5 L of a 1:8 dilution; Lane 9, untreated human serum, 5 L of a 1:80 dilution. Before Coomassie staining caused a higher variability of quantitation, which rendered a coefficient of variation (CV) of 19.7%, versus a CV of only 7.8% using the Criterion Stain Free Imaging System. %%EOF
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Figure 2. The Criterion Stain Free Imaging System eliminates the need to run and Coomassie stain a duplicate gel to assess the protein pattern before transfer to the membrane as well as the need for time- and reagent-consuming traditional methods such as Ponceau S staining to assess protein transfer prior to detection in Western blotting. New Imaging Technique with Broad Utility Provides Protein Assessment Alternative. 0000012499 00000 n
Sundaram RK, Balasubramaniyan N, Sundaram P. Methods Mol Biol. , SDS-PAGEVolker NeuhoffKumasiCoomassie, R-250G-2502R-250G-250, DNRGelCapture, 465 nm 595 nm, 470nmLEDUV() F-ChemiBIS 6 Pro Bio-Imaging , DNRF-ChemiBIS 6 Pro SDS PAGE4 15 -R B8647 E.Coli MEK1 , 131-00462-8-12Tel03-6661-8064Fax03-6661-8065, 532-00113-11-102Tel06-6195-8750Fax06-6195-8760. Federal government websites often end in .gov or .mil. HHS Vulnerability Disclosure, Help The .gov means its official. government site. Variables in Coomassie staining/destaining conditions such as agitation times, volumes, solution changes, and temperature affect the reproducibility of results. Muntoni E, Martina K, Marini E, Giorgis M, Lazzarato L, Salaroglio IC, Riganti C, Lanotte M, Battaglia L. Pharmaceutics. Traditional methods of protein gel detection and imaging have been improved and expanded through technological advancement. %PDF-1.6
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Coomassie staining has its drawbacks, however. }m iYhprL@#8$4nl)DZw Since tryptophan is typically between only 1 and 2% of the amino acid residues in most proteins, most antibodies will not be affected by Trp modification of the target proteins. Extending this analysis across all of the bands in the protein standard, the Criterion Stain Free Imaging System demonstrated superior or comparable limits of detection (LODs; 0.2 to 5 ng) and limits of quantitation (LOQs; 0.5 to 6 ng) for all of the proteins except aprotonin, which does not contain tryptophan (Figure 1). 45 0 obj <>
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Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". 0000006530 00000 n
All Rights Reserved. 0000006056 00000 n
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Figure 1. Monoclonal antibodies can also be used if they are not specific for an epitope containing tryptophan. The gels were activated and imaged on the Criterion Stain Free system (A), then stained with Bio-Safe G-250 stain (B), followed by imaging on a Molecular Imager GS-800 calibrated densitometer. It has been demonstrated that UV activation does not interfere with the transfer or immunodetection of two human proteins when using polyclonal antibodies. The https:// ensures that you are connecting to the Processes such as quality control of therapeutic proteins and monitoring of protein expression rely on reproducible quantitation of protein PAGE bands, which in turn relies on the quality of the method used to visualize the protein. trailer
sharing sensitive information, make sure youre on a federal Bethesda, MD 20894, Web Policies The utility of this novel visualization technique will be judged largely on its compatibility with techniques like Western blotting and mass spectrometry. Today, the detection of proteins, resolved on gels, can be accomplished with a variety of stains with various sensitivities. These rates of identification are typical of MS analyses of PAGE. Methotrexate-Loaded Solid Lipid Nanoparticles: Protein Functionalization to Improve Brain Biodistribution. 0000031483 00000 n
Forty-six spots gave the same identities as those extracted from nonirradiated gels. 0000000016 00000 n
Coomassie-Brilliant Blue staining of polyacrylamide gels. Glycoproteins, which make up more than half of all proteins, stain poorly with Coomassie dye, and there is at least one report in the literature that Coomassie staining may overstate relative protein quantities in gels. The Criterion Stain Free Imaging System can be used with a variety of complex protein samples to form a number of sources, with the results being visually equivalent to Coomassie staining. Kevin McDonald, Ph.D. (kevin_mcdonald@bio-rad.com), is senior research scientist at Bio-Rad Laboratories. Another major advantage of the system is that it lends itself well to automation: the gel can be activated, the digital image captured, and the molecular weight and quantity of each protein band can be calculated by an automated system with the push of a button. Disclaimer, National Library of Medicine 2016 Oct 11;7(41):66679-66688. doi: 10.18632/oncotarget.11439. xref
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Bookshelf SDS-PAGE followed by Coomassie staining is a standard, widely used method to visualize proteins. After electrophoresis, the gel is subjected to UV irradiation for as little as 2.5 minutes, which activates a covalent reaction between the trihalocompound and tryptophan residues on the proteins in the gel. In contrast, using the Criterion Stain Free Imaging System, gels have a uniform and low background level and yield consistent and reproducible results. The resultant adduct of tryptophan is fluorescent when excited by the same UV source. Methods Mol Biol. Arndt C, Koristka S, Feldmann A, Bartsch H, Bachmann M. Methods Mol Biol. 0000009653 00000 n
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Limits of detection (LOD) and limits of quantitation (LOQ) of proteins using either UV activation imaging or Coomassie Blue G-250 staining: Individual protein bands from a broad range of protein molecular weight standards visualized with UV activation (420% Criterion Stain Free gel) and subsequently with Coomassie G-250 stain (Bio-Safe) from four replicate gels were used to determine visual LOD and LOQ. 2012;869:465-9. doi: 10.1007/978-1-61779-821-4_40. An official website of the United States government. This site needs JavaScript to work properly. The relative low cost of these dyes and their ready-made solutions, sensitivity in the 550 ng range, and time-tested staining and destaining protocols have been keys to their wide acceptance. MeSH Would you like email updates of new search results? 0000001055 00000 n
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Copyright 2022 Genetic Engineering & Biotechnology News. Careers. Life Sci. In addition to allowing rapid and direct confirmation of appropriate protein patterns, this technique enables efficient transfer of protein bands to membranes and subsequent immunodetection (Figure 2). To compare the reproducibility of quantitation using Coomassie staining versus the Criterion Stain Free Imaging System, serial dilutions of a PAGE protein standard were run on polyacrylamide gels in quadruplicate, visualized with UV irradiation, and subsequently stained with Coomassie G-250. A database search with a dynamic modification of the tryptophan residue helped to assure identification of all peptides containing trp, and may assist in the identification of low-abundance proteins. endstream
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Here we describe the UVP BioImaging System in combination with LabWorks Image and Acquisition software to provide a comparison of four different protein gel stains: Lumitein, ProteoSilver, SYPRO() Ruby, and Coomassie() Brilliant Blue. A brief review of other notable protein detection methods on acrylamide gels. 0000032733 00000 n
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Clipboard, Search History, and several other advanced features are temporarily unavailable. Coomassie is also known to vary widely in its ability to bind proteins, due at least in part, to its affinity for proteins rich in basic amino acids such as arginine, histidine, and lysine. 2012;869:585-9. doi: 10.1007/978-1-61779-821-4_53. 0000009839 00000 n
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Finally, Coomassie staining can generate large quantities of solvents, some of which are hazardous. Rapid detection of proteins in polyacrylamide electrophoresis gels with Direct Red 81 and Amido Black. An old method facing a new challenge: re-visiting housekeeping proteins as internal reference control for neuroscience research. <<41EFA716EEE6C0489ACDB2D3673882B4>]>>
The entire electrophoretic separation and gel imaging can be completed in about an hour.
In most organisms, proteins without tryptophan represent less than 10% of the proteins from 10260 kD, the separable range for most PAGE gels. $d1H0%u=NWl?FI 0000025125 00000 n
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Bio-Rad Laboratories Criterion Stain Free Imaging System uses a trihalocompound modification of tryptophan providing a desirable alternative to Coomassie staining for many applications. The bands detected on the gel by the Criterion Stain Free Imaging System can subsequently be transferred to membranes, while bands stained with Coomassie typically cannot. 8600 Rockville Pike PMC [Protein determination by binding with the dye Coomassie brilliant blue G-250]. 2012;869:451-64. doi: 10.1007/978-1-61779-821-4_39. Gel staining and destaining with Coomassie can take at least two hours. Methods Mol Biol. The quantity of protein present in the b-galactosidase band (MW 116,000) was estimated using Bio-Rads Image Lab automated image-analysis software. Uneven staining of the gels may result in erroneous quantitation of the protein bands. Epub 2013 Feb 27. 2019 Feb 2;11(2):65. doi: 10.3390/pharmaceutics11020065. 2012;869:617-20. doi: 10.1007/978-1-61779-821-4_56. 81 0 obj<>stream
The fluorescent signal is then automatically imaged in less than a few seconds by the Criterion Stain Free Imaging System, which produces an image of proteins in the gel. 0000003897 00000 n
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GEN Genetic Engineering and Biotechnology News, How Intestinal Gut Cells Communicate With and Promote Survival of T, Human Brains Functional Connectivity Boils Down to Three Patterns in Time, Beneficial Biofilms Thrive on New Synthetic Polymers, Plant-Based Meat Benefits People and the Planet, Rare Mutations in CIDEB Gene Protect against Liver Disease, Outlining the Latest Regulatory Trends in Advanced Therapies, Like Cell and, Molecular Code for Lewis X Makes Glycosylation Controllable, COVID-19 Drives Surge in Growth of Single-use Technologies, Next-Generation Sequencing Moves to Next-Next Level, Santhera Confirms Plans to File Catena for Treating LHON Eye Disease, Spatial and NGS Dominate Proceedings at AGBT, Anti-Inflammatory Drugs Demonstrate Pathway Savvy, How Intestinal Gut Cells Communicate With and Promote Survival of T Cell Guardians, Human Brains Functional Connectivity Boils Down to Three Patterns in Time and Space, Opportunities & Challenges in Latin America. The limits of detection and signal intensities are the same, with or without UV irradiation of the gels. o /5)ak)_>^}]&eHdc=g.R8&AG406Kl#h\fPr74?X[ q!j)n
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We demonstrate that the detection sensitivity limit appears to be between 100 and 500 ng/protein band of protein with Coomassie() Brilliant Blue, 10-50 ng/protein band with Lumitein and SYPRO() Ruby, and as little as 5 ng/protein band with the ProteoSilver stain. Samples were run on a 420% Criterion Stain Free Tris-HCl gel (Bio-Rad Laboratories) containing the trihalocompound. 0000006612 00000 n
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Hm4d'g. 2013 Apr 19;92(13):747-51. doi: 10.1016/j.lfs.2013.02.014. 0000001501 00000 n
The UV-induced modification of tryptophan residues in gel protein bands also does not interfere with subsequent mass spectrometric identification of the proteins. 0000030909 00000 n
Please enable it to take advantage of the complete set of features! The system uses standard sample preparation, reagents, and electrophoresis protocols, with the trihalocompound incorporated into standard gel formulations. Additionally, this imaging method improves the ease of use of Western blotting while maintaining sensitivity of detection, and can be used with MS techniques for protein identification. The site is secure. Accessibility Hu X, Du S, Yu J, Yang X, Yang C, Zhou D, Wang Q, Qin S, Yan X, He L, Han D, Wan C. Oncotarget. 0000032455 00000 n
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A large-scale study of a total of 56 spots from two sets of UV-activated 2-D gels resulted in identification of 48, using LC-MS/MS analysis, MALDI-TOF-MS analysis, or both. Images of lanes of blue bands are universally recognized in the life science research community as PAGE protein separations. FOIA 0000001366 00000 n
The Criterion Stain Free Imaging System enables quick and easy monitoring of the efficiency of transfer by imaging the membrane and gel after transfer. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins can be visualized by various methods of detection and imaging. official website and that any information you provide is encrypted Examination of predicted proteomes for common experimental organisms shows that proteins lacking tryptophan are biased toward small molecular weight. Unable to load your collection due to an error, Unable to load your delegates due to an error. The disadvantages that accompany Coomassie staining can be cured through the use of a unique protein visualization technique that eliminates two hours of processing time, provides equivalent or superior sensitivity and reproducibility of quantitation, and is suitable for analysis of a wide range of complex protein samples. 0000012280 00000 n
Digital cameras used in the imaging of protein gels are not only more sensitive than their film precursors, but they can be used in combination with imaging software that offers a host of useful applications. In fact, the percentage of human proteins larger than 10 kD that lack tryptophan is only 7.3%. 0000017390 00000 n