...RFP fluorescence suggests an increase in autophagic flux, while an increase in GFP fluorescence relative...of several fluorescent autophagy reporters now allows for the measurement of autophagic flux, or the changes... to emission of only red fluorescence. The ratio of red to green fluorescence allows for clear distinction...control. Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP...relative to RFP fluorescence suggests a decrease in autophagic flux.   Find the GFP-LC3-RFP-LC3∆G plasmid at...photoconversion Light sheet fluorescence microscopy Resources on Addgene.org Fluorescent protein plasmids and...autophagy biosensor that’s comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant...

... of fluorescently-labeled components Fluorescence-activated cell sorting (for cells); fluorescent HPLC...and fluorescent reporters being especially popular. We’ll give you an introduction to fluorescent biosensors...calcium and zinc. Some biosensors contain a single fluorescent protein fused to a domain that senses the concentration...When calcium is absent, the construct does not fluoresce; when present, calcium induces CaM/RS20 interaction...s structural organization and activating the fluorescent signal. Blue flame GECO biosensors from Robert...s lab display calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors are another...sensors created by Roger Tsien’s lab contain two fluorescent proteins (a FRET pair) separated by CaM and M13...

... as fluorophores in several different ways. Researchers have used oligonucleotides in fluorescent visualization...decades, most notably for fluorescent in situ hybridization (FISH) where fluorophore-linked antibodies are...applications including aptamer based fluorescent biosensors. Aptabeacons Fluorescence resonance energy transfer ...state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore and quencher...aptamers fluoresce until they bind to the appropriate ligand, but relying on a lack of fluorescence for readout...applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing with veggies) Though the...is bound to a protein fluor. Transcribing and translating a protein fluorophore also takes time - bypassing...

..., like luciferases, as well as fluorescent ones, like Green Fluorescent Protein (GFP). These molecules...previously reach. Nobel Prize for fluorescent proteins The importance of fluorescent proteins was recognized in...learning more about fluorescent proteins, I invite you to download our Fluorescent Protein eBook and learn...laboratory. The discovery of GFP The story of how fluorescent proteins like GFP came to be used as research...function and shift its emission spectrum. New fluorescent proteins with new colors Around the same time...hunch paid off, and he was able to report new fluorophores like DsRed from Discosoma sp. and Katushka/mKate...quadricolor. With his discoveries, another part of the fluorescent protein puzzle fell into place, as they, taken...

...time.Hallmarks of fluorescent protein timers When FP timers are first expressed, they predominantly fluoresce one ...independently) originally fluoresce green or blue and, when they mature, fluoresce red. The ratio of the ...Advice on Choosing Your Fluorescent Protein Resources at Addgene.org Find Fluorescent Protein Timers Browse...Browse All Fluorescent Protein Empty Backbones Check Out Our Fluorescent Protein Guide ...Even before fluorescent proteins (FPs) came into wide use, there were a variety of ways to monitor cell...situ hybridization experiments. In many cases fluorescent proteins have usurped old methods or complemented...color but slowly mature until they predominantly fluoresce a second color. This change is usually hypothesized...

...from low fluorescent states to high fluorescence states, they can change from one fluorescent color to...proteins. Photoactivatable fluorescent proteins (PA-FPs) are fluorescent proteins that display unique...their fluorescence can be switched on and off reversibly. This ability to modulate fluorescence allows...switched on from a state of dim or no fluorescence, to a brighter fluorescent state by exposure to a specific...photoconvertible fluorescent proteins (PC-FPs). PC-FPs can convert from one fluorescent color to another... a bright fluorescent state (“kindling”), and from a bright state to a dark non-fluorescent state (“quenching...reversible, monomeric red fluorescent PA-FPs. rsCherries have a high background fluorescence, but their single...

...Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed...iterations of fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins...specific mutations comprising common fluorophores Fluorescent Protein Mutations Relative to Wildtype...folding, maturation, and fluorescence. Biosensors: A wide array of GFP-based fluorescent biosensors has been...techniques in fluorescent microscopy. Read More: All Plasmids 101 Articles Addgene's Fluorescent Protein ... its biochemical properties. Now GFP and its fluorescent derivatives are a staple in the lab. GFP is used... common applications in the lab. Why Green Fluorescent Protein? GFP is a ~27 kDa protein consisting ...

...opportunities. Xin Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied...Brightest Fluorescent Protein for Your Experiments Resources on Addgene.org Find Fluorescent Protein ...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable...applications, researchers are finding new ways to apply fluorescent proteins everyday.  The late Michael Davidson... and develop these tools by making their own fluorescent protein technologies widely available to the ... are intended to help scientists share their fluorescent protein research with the academic community ...stood out for their creativity in the use of fluorescent proteins and the potential for their work to ...

...track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a fluorescence microscope...for many fluorescent proteins. Additional fluorescent protein tools Photoactivable fluorescent proteins...the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter... By using cells labelled by fluorescent chemicals or by other fluorescent proteins, labs were also able... that exist in vivo. The use of fluorescent pathogens and fluorescent cells enables scientists to monitor...Laser include: Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, ...irreversible photoconversion from green fluorescence to red fluorescence) are also useful tools to use with...

...turn up the brightness in your fluorescent imaging experiments. Fluorescent protein fusions One of the easiest...Blog Sign Up to Receive the Fluorescent Protein eBook Avoid Fluorescent Protein Oligomerization Learn...Learn about Small Molecule Fluorphores Resources on Addgene.org Find Fluorescent Protein Empy Backbones ...biological lesson and created a system to amplify fluorescent signals. Named for the "stellar explosion SUperNova... to track a given protein is to fuse it to a fluorescent protein. You can then study where the protein...these problems? Instead of directly fusing a fluorescent protein to your protein of interest, you instead.... This system amplifies the intensity of the fluorescent signal and enables single molecule tracking  ...

...the fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified...small molecule activates the fluorescence of the small molecule. Fluorescent RNA aptamers are used to track... applications of fluorescent RNA aptamers in Addgene’s collection. Green fluorescent aptamers Spinach ...selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution...blog Learn more about aptamer fluorophores Investigate other fluorescent biosensors Find other uses for...using aptamers might know this best, as most fluorescent aptamers are named for a fruit or vegetable! ...What is an RNA aptamer? RNA is not inherently fluorescent and can be challenging to track intracellularly...

...reagents like fluorescently-labeled nanobodies or scFvs. Many, many options for fluorescently conjugated ...then label that with fluorescently-conjugated streptavidin to get more fluorophores per antibody, or conjugate...Antibodies Which Fluorescence Microscopy Technique is Best for Me? Introduction to Immunofluorescence Resources...You may be familiar with immunofluorescence (IF, often referred to as immunocytochemistry (ICC) when ...binds a target protein in your sample, then a fluorescently labeled secondary antibody binds that first ...The general process is similar to standard immunofluorescence, but with some extra considerations (Im et...  Figure 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where...

... using fluorescence (for some background on fluorescence, try the Introduction to Fluorescence Microscopy...proteins. You can then use fluorescent microscopy to look for those fluorescent molecules, knowing that ...to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence and indirect...Introduction to Fluorescence Microscopy. iBiology. https://www.ibiology.org/talks/fluorescence-microscopy/...Antibodies Learn more about fluorescence microscopy in Which Fluorescence Microscopy Techniques is Best...Immunofluorescence (IF), is an immunoassay that brings to light the cellular world. The technique allows...and performing an IF experiment. What is immunofluorescence? Generally speaking, IF is an application...

...Developments in light sheet fluorescence microscopy Modern light sheet fluorescence microscopy was first pioneered... advances in microscopy has been light sheet fluorescence microscopy (LSFM). The underlying method was...colleagues and originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David...laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected light using...configurations’ various features see (4,7).   Light sheet fluorescence microscopy vs. confocal microscopy In confocal...However, the excitation light excites all of the fluorophores as it passes through the specimen, which often...obstacles (e.g. air bubbles or high concentration of fluorophores) cast shadows along the illumination path. The...

...occurs slower than fluorescent-based methods, such as excitation of Green Fluorescent Protein (GFP) because...luminescence versus fluorescence.  Table 1: Properties of Luminescence versus Fluorescence   Luminescence...Learn How Luciferase Can Be Used In Concert with Fluorecent Proteins in Nano Lanterns Reporter gene assays...chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage of lower background...Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon...

...in a change in the fluorescent readout. In this blog we will focus on two fluorescent CRISPR reporter methods...surpass GEmCherry2 in background fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry...assays. Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage...enrich for mutants through the presence of a fluorescent reporter. Lastly, once a mutant is identified...at Addgene GEMCherry2, an improved monomeric fluorescent protein for assessing CRISPR/Cas9 activity Guide...but most are labor-intensive, require multiple fluorescent proteins, and aren’t that efficient. To overcome...Denham lab developed a modified monomeric red fluorescent protein (RFP), named GEMCherry2, that can rapidly...

... months ago, we shared an introduction to immunofluorescence (IF) - a common method for visualizing molecules...1985) Postfixation detergent treatment for immunofluorescence suppresses localization of some integral ...Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. Methods in Molecular Biology. Springer...Addgene blog: Antibodies 101: Introduction to Immunofluorescence Antibodies 101: Epitope Availability Antibodies...

...emission by fluorescence. The widespread use of FRET with fluorescent molecules, including fluorescent proteins...donor fluorophore to the acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation...acceptor fluorophore--think of it as a virtual photon 3. Relaxation of the acceptor fluorophore back to...acceptor fluorophores must be compatible, such that the emission spectrum of the donor fluorophore overlaps..., et al. "Fluorescent protein based FRET pairs with improved dynamic range for fluorescence lifetime measurements...Miyawaki, Atsushi, et al. "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin... Out Fluorescent Protein Biosensors Browse the Entire Fluorescent Protein Collection ...

... is the permanent fluorescent tag portion of Rosella: it will emit red fluorescence regardless of its ...the fluorescent properties of Rosella.   Table 2: Effect of Localization on Rosella’s fluorescence emission...green fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks ...Photoconversion Light Sheet Fluorescence Microscopy Additional Resources on Addgene.org Fluorescent Protein Plasmids...Plasmids and Resources Fluorescent Protein Guide: Biosensors Keima, another fluorescent pH-biosensor for studying...Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark ...Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a...

...containing several types of fluorescent (EGFP, tagRFP, mCherry) and nonfluorescent (Halo, SNAP) tags, a selectable...Blog Learn about Fluorescent Protein Timers Check out Photoactivatable Fluorescent Proteins Use Rosella...SapTrap Kit Page Find Other Fluorescent Protein Kits Browse the Fluorescent Protein Collection ...GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has been an invaluable resource...limitations to working with FPs. Overexpression of fluorescently tagged proteins can lead to improper protein...One way to avoid the pitfalls of overexpressed fluorescent protein fusions is to replace the genomic copy...copy of your gene of interest with a fluorescent protein fusion. CRISPR genome editing can help accomplish...