{"id":9827,"date":"2023-02-07T06:06:52","date_gmt":"2023-02-07T11:06:52","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=9827"},"modified":"2024-12-02T07:11:06","modified_gmt":"2024-12-02T12:11:06","slug":"fluorescent-molecules","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/company-news\/fluorescent-molecules\/","title":{"rendered":"Fluorescent molecules"},"content":{"rendered":"\n\n\n\t<div class=\"dkpdf-button-container\" style=\" text-align:right \">\n\n\t\t<a class=\"dkpdf-button\" href=\"\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9827?pdf=9827\" target=\"_blank\"><span class=\"dkpdf-button-icon\"><i class=\"fa fa-file-pdf-o\"><\/i><\/span> Download PDF<\/a>\n\n\t<\/div>\n\n\n\n\n\n<p><style>.entry p, .entry ol{font-size: 1rem;}.entry h3{color:#009fe3;margin-top:0}.entry h4{color:#009453;margin-top:1.5rem}.entry h5{color: #003a5c; font-size: 1.05rem;margin-top:1.5rem;}.entry figure{margin:32px auto;border:1px solid #ccc}.entry figure img{width:100%;display:block;margin:0 auto}.entry figcaption{font-size:.875rem;line-height:1.35rem;padding:10px;color:#222}.entry .blog-tbl{margin:1rem auto;caption-side:bottom}.entry .blog-tbl td,.entry .blog-tbl th{padding:10px 9px;border:1px solid #00172b;text-align:left}.entry .blog-tbl td{border-color:#003a5c}.entry .blog-tbl th{background-color:#00172b;color:#fff;border-left-color:#fff;border-right-color:#fff}.entry .blog-tbl th p{color:#fff}.entry .blog-tbl th:first-of-type{border-left-color:#00172b}.entry .blog-tbl th:last-of-type{border-right-color:#00172b}.entry .blog-tbl p{text-align:left;margin:0}.entry .box-note{border:2px solid #009453;padding:12px;margin:1rem 0}.entry .box-note p{margin:0;padding:0}.entry .styled-list{list-style-type:none}.entry .styled-list li{margin-top:1rem;line-height:1rem;font-size:1rem;}.entry .styled-list li::before{font-family:\"Font Awesome 5 Pro\";display:inline-block;content:\"\\f3c5\";-webkit-transform:rotate(-90deg);transform:rotate(-90deg);margin-left:-20px;margin-right:11px;font-size:.75rem;color:#ed7004;font-weight:600}.entry .styled-list ol li::before{display:none}.entry .styled-list li>ul li::before{font-weight:200}.entry .overview{width:-webkit-fit-content;width:-moz-fit-content;width:fit-content;padding:16px;margin:1rem auto;border:1px solid #eee}.entry .overview hr{margin-top:14px}.entry .overview-text{text-align:center;font-size:1rem;margin:0}.entry .btn-sq{color:#fff;font-size:.9rem;font-weight:600;border:2px solid rgb(237, 112, 4);padding:7px 13px;background:rgb(237, 112, 4);cursor:pointer;text-align:center;line-height:1.5rem;margin:0 auto;}.entry .btn-sq:hover{text-decoration:none;color:rgb(237, 112, 4);background:#fff;}.entry .btn-container{display:flex;width:100%;margin:1.3rem 0;}.entry .btn-container .fa-solid, .entry .btn-container .fa-duotone{margin-right:10px;}@media(max-width: 768px){.entry .btn-sq{font-size:1.05rem;}}<\/style>\n<style>.entry .styled-list li::before{display:none;} .entry .styled-list{list-style-type:decimal}.entry .styled-list li::marker{font-size:75%}.entry ol li {line-height:24px;margin-bottom:.4rem}<\/style>\n<\/p>\n<p><script src=\"https:\/\/kit.fontawesome.com\/904923013f.js\" crossorigin=\"anonymous\"><\/script><\/p>\n<h2>Fluorophores are essential to many different applications, from flow-cytometry, confocal and super-resolution microscopy to Western blotting and ELISA. From recombinant fluorescent proteins to the hugely popular fluorescent organic dyes when conjugated with secondary antibodies, they enable the visualization of the target antigen. Here we explore the history and progress of the application of fluorescent molecules in science.<\/h2>\n<p><!--more--><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-9830 \" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro.jpg\" alt=\"\" width=\"817\" height=\"174\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro.jpg 512w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro-300x64.jpg 300w\" sizes=\"(max-width: 817px) 100vw, 817px\" \/><\/p>\n<h4>Fluorescent proteins<\/h4>\n<p><span style=\"font-weight: 400;\">Although the discovery of bioluminescence dates back to the 1500s, it wasn\u2019t until 1852 that the term &#8220;fluorescence&#8221; came into use. &#8220;Fluorescence&#8221; was coined by George Gabriel Stokes (Web ref:1, Kalyuzhny 2016) after discovering that the mineral fluorite emitted visible light after exposure to ultraviolet light.&nbsp; A century later, in 1955, Green Fluorescent protein (GFP) was identified in the Jellyfish <\/span><i><span style=\"font-weight: 400;\">Aequorea victoria <\/span><\/i><span style=\"font-weight: 400;\">by Davenport &amp; Nichol (2), and later isolated by Osama Shimomura in 1962. However, it wasn\u2019t until 1996, when Roger Tsien&#8217;s group identified a point mutation in GFP (S65T), making the protein practical for research applications, that GFP\u2019s research possibilities were realized (3).<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Since then, with optimization, the recombinant protein GFP has been modified to yield many variations in color, from cyan and the original green through to the reds of mCherry and mTomato, with improvements in photostability, brightness, and experimental vigor.<\/span><\/p>\n<p><span style=\"font-weight: 400;\">GFP provided a starting block for the study of biological processes in living cells and the development of techniques that wouldn\u2019t be possible without the unique properties of color. The impact of GFP on the world of research was highlighted by the award of the Nobel Prize for Chemistry in 2008 to Shimomura, Tsien, and Chalfie for their work on GFP (4).<\/span><\/p>\n<p><span style=\"font-weight: 400;\">GFP and its analogs can be \u201ccoded\u201d into the protein sequence and \u201cexpressed\u201d as part of the protein at points deemed appropriate based on structure and known or predicted function. Expression of GFP fusion proteins can present challenges that can limit their use. Although they may enable experiments such as co-localization or even F\u00f6rster Resonance Energy Transfer (FRET), allowing stoichiometrical characterization, such experiments require careful development.<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-703\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/gfp-1ema-chromo.jpg\" alt=\"1EMA structure of GFP with Chromophore\" width=\"700\" height=\"293\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/gfp-1ema-chromo.jpg 1747w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/gfp-1ema-chromo-300x126.jpg 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/gfp-1ema-chromo-1024x429.jpg 1024w\" sizes=\"(max-width: 700px) 100vw, 700px\" \/><figcaption><strong>Figure 1:<\/strong> The crystal structure of GFP (A + B) shows how the beta-barrel protects the chromophore (C) within. (PDB code 1EMA) (5)<\/figcaption><\/figure>\n<p><span style=\"font-weight: 400;\">Transfection of plasmids coding for the fluorescent protein and the gene of interest can be time-consuming, requiring optimization, and the fluorophore may change the behavior of the protein of interest, negating the value of the experiment.&nbsp;<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Fluorescence, however, was a captivating characteristic that, if harnessed, the potential value of which scientists couldn\u2019t ignore. This led researchers to develop other methods to add fluorophores to their molecules of interest without the artifacts incurred by expressing a recombinant fluorescent protein.<\/span><\/p>\n<hr>\n<h4>Organic dye and fluorescent protein conjugates<\/h4>\n<p><span style=\"font-weight: 400;\">The antigen:antibody interaction is a tractable way of labeling proteins <\/span><i><span style=\"font-weight: 400;\">in situ<\/span><\/i><span style=\"font-weight: 400;\"> on live cells, as well as adding multiple fluorescent molecules per antigen\u2013amplifying signal. Fluorescent molecules suitable for labeling have been developed from the phycobiliproteins of photosynthetic algae (8) to the much smaller organic dyes like Alexa Fluor\u00ae and Cyanine dyes,&nbsp; and much smaller fluorescent particles like Quantum dots. These other molecules have increased the range of colors available to scientists and have enhanced the qualities of the fluorophores available, allowing their application to be diversified into numerous techniques.<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Organic dyes and fluorescent proteins can be used as fluorescent probes conjugated (or \u201ctagged\u201d) to antibodies, allowing the researcher to take advantage of many of the benefits of <\/span><i><span style=\"font-weight: 400;\">in situ<\/span><\/i><span style=\"font-weight: 400;\"> staining over recombinant expression of fluorophores.<\/span><\/p>\n<p><span style=\"font-weight: 400;\">The first conjugated fluorophores to appear were FITC (fluorescein isothiocyanate) and TRITC (tetramethyl rhodamine isothiocyanate) (7) back in the 1950s. As computing power has increased to cope with the processing demands of multi-channel experiments and our optical techniques have developed, the range of available dyes has exploded.<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-9828 \" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/dye-molecules.jpg\" alt=\"\" width=\"669\" height=\"251\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/dye-molecules.jpg 512w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/dye-molecules-300x113.jpg 300w\" sizes=\"(max-width: 669px) 100vw, 669px\" \/><p><\/p>\n<figcaption><strong>Figure 2:<\/strong> Alexa Fluor\u00ae 488 and 594 dye molecules (6).<\/figcaption>\n<\/figure>\n<p><span style=\"font-weight: 400;\">Conjugated fluorescent probes offer numerous advantages to recombinant proteins. They can have less potential for steric interference and expression artifacts, as well as offer the signal enhancement benefits of indirect detection &#8211; by binding more dye molecules per antigen molecule &#8211; potentially increasing the brightness &#8211; adding sensitivity to the assay. The antibody conjugates can also offer improved photostability, decreased photobleaching, and a more \u201cuser-friendly\u201d format. Numerous fluorescent conjugates are available, spanning the spectrum from blue through to infrared.<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Jackson ImmunoResearch offers a comprehensive collection of conjugates spanning the entire spectrum. Phycobiliproteins &#8211; Phycoerythrin (PE), Peridinin chlorophyll protein (PerCP), and Allophycocyanin (APC), Alexa Fluor\u00ae, Brilliant Violet, and Cyanine dyes conjugated to Jackson Immunoresearch\u2019s secondary antibodies offer a kaleidoscope of possibility.<\/span><\/p>\n<hr>\n<h4>References:<\/h4>\n<ol>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">https:\/\/www.britannica.com\/biography\/Sir-George-Gabriel-Stokes-1st-Baronet<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">Davenport D, Nicol JAC. (1955). Luminescence in Hydromedusae. Proc. R. Soc. Lond. B 144, 399\u2013411.<\/span><\/li>\n<li aria-level=\"1\">\n<p><span style=\"font-weight: 400;\">SHIMOMURA, O., JOHNSON, F. H., &amp; SAIGA, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. <\/span><i><span style=\"font-weight: 400;\">Journal of cellular and comparative physiology<\/span><\/i><span style=\"font-weight: 400;\">, <\/span><i><span style=\"font-weight: 400;\">59<\/span><\/i><span style=\"font-weight: 400;\">, 223\u2013239. <\/span><a href=\"https:\/\/doi.org\/10.1002\/jcp.1030590302\"><span style=\"font-weight: 400;\">https:\/\/doi.org\/10.1002\/jcp.1030590302<\/span><\/a><\/p>\n<\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">Heim R, Tsien RY. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. <\/span><span style=\"font-weight: 400;\">Current Biology<\/span><span style=\"font-weight: 400;\">. 1996 Feb 1;6(2):178-82.<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">&nbsp;Roger Y. Tsien<\/span><span style=\"font-weight: 400;\"> (2010) The 2009 Lindau Nobel Laureate Meeting: Roger Y. Tsien, Chemistry 2008. <\/span><span style=\"font-weight: 400;\">J Vis Exp<\/span><span style=\"font-weight: 400;\">. 2010; (35): 1575.<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">&nbsp;Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., Remington, S.J.(1996) Crystal structure of the <\/span><i><span style=\"font-weight: 400;\">Aequorea victoria<\/span><\/i><span style=\"font-weight: 400;\"> green fluorescent protein. <\/span><span style=\"font-weight: 400;\">Science<\/span> <b>273<\/b><span style=\"font-weight: 400;\">: 1392-1395<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">Nataliya Panchuk-Voloshina et al. Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates. <\/span><span style=\"font-weight: 400;\">J Histochem Cytochem<\/span><span style=\"font-weight: 400;\"> 1999;47:1179-1188<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">RIGGS JL, SEIWALD RJ, BURCKHALTER JH, DOWNS CM, METCALF TG. (1958). Isothiocyanate compounds as fluorescent labeling agents for immune serum. <\/span><span style=\"font-weight: 400;\">Am J Pathol<\/span><span style=\"font-weight: 400;\">. 1958 Nov-Dec;34(6):1081\u20131097.<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">COONS AH, KAPLAN MH. (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. <\/span><span style=\"font-weight: 400;\">J Exp Med<\/span><span style=\"font-weight: 400;\">. 1950 Jan 1;91(1):1-13.<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">Morisset. K &amp; Walter.B (1984), Phycobiliproteins \u2014 characterization of coloured algal proteins by a simple electrophoretic procedure. <\/span><span style=\"font-weight: 400;\">Biochemical Education<\/span><span style=\"font-weight: 400;\"> (12.4).<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400;\">&nbsp;Kalyuzhany (2016) Immunohistochemistry &#8211; Essential elements and beyond. Springer. Switzerland<\/span><\/li>\n<\/ol>\n<p><a href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/AffiniPure-VHH-secondary-antibodies\"><img decoding=\"async\" style=\"width: 100%;\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Nano-Secondaries-Advert-ML-GIFFING-4.gif\"><\/a><\/p>\n<table class=\"table blogLinks\">\n<thead>\n<tr>\n<th class=\"span6\">Learn more:<\/th>\n<th class=\"span6\">Do more:<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/directandindirectwesternblotting\/\">Indirect and direct Western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/exhibitions\">Exhibition Schedule<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/chemiluminescent-western-blotting\/\">Chemiluminescence western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/offers\/citeandwin\">Cite and Win<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a class=\"row-title\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-admin\/post.php?post=3642&amp;action=edit\" aria-label=\"\u201cAn Introduction to Expansion Microscopy\u201d (Edit)\">An Introduction to Expansion Microscopy<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/offers\/previous-winners\">Past Competition Winners<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n<p><\/p>\n<!-- AddThis Advanced Settings generic via filter on the_content --><!-- AddThis Share Buttons generic via filter on the_content --><!-- AddThis Related Posts generic via filter on the_content -->","protected":false},"excerpt":{"rendered":"<p>Download PDF Fluorophores are essential to many different applications, from flow-cytometry, confocal and super-resolution microscopy to Western blotting and ELISA. From recombinant fluorescent proteins to the hugely popular fluorescent organic dyes when conjugated with secondary antibodies, they enable the visualization of the target antigen. Here we explore the history and progress of the application of [&hellip;]<!-- AddThis Advanced Settings generic via filter on get_the_excerpt --><!-- AddThis Share Buttons generic via filter on get_the_excerpt --><!-- AddThis Related Posts generic via filter on get_the_excerpt --><\/p>\n","protected":false},"author":3,"featured_media":9830,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"content-type":""},"categories":[19,20],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9827"}],"collection":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/comments?post=9827"}],"version-history":[{"count":15,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9827\/revisions"}],"predecessor-version":[{"id":11112,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9827\/revisions\/11112"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media\/9830"}],"wp:attachment":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media?parent=9827"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/categories?post=9827"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/tags?post=9827"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}