...SDS-PAGE gels containing Phos-Tag, a small molecule that binds specifically to phosphate moieties and promotes... al. "Detection of phosphorylation on large proteins by Western blotting using Phos-tag containing gel...MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic dual...shift in phosphorylated, but not non-phosphorylated proteins (8). Quantifying the ratio of phospho- and non-phospho-bands...of the dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the...

...or dark conditions, the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription...histidine kinase/photosensor senses various light inputs, and an obligate chromophore allows the system...dark conditions, the chromophore activates the response regulator via phosphorylation, promoting transcription...in E. coli easier and more robust. Synthetic photobiology: why use light? In synthetic biology and bioengineering...TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses...two-component system (TCS). Levskaya et al. fused a photosensory domain from cyanobacteria to a common E. coli...hybrid construct senses light, and an obligate chromophore allows the system to respond to various light...

...dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and pa= photoactivation) domains on... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to the...-specifically activated and, like the game of telephone, often the signal must be transmitted by intermediate... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is...

...state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore and quencher...of aptamers means they can be used with or as fluorophores in several different ways. Researchers have ...fluorescent in situ hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. ...removing the quencher from the proximity of the fluorophore and subsequently leading to a positive signal...signal. The same effect can be achieved for a fluorophore that relies on dimerization for activation, only...used as powerful biosensors, the bound protein fluorophores and quenchers are still relatively bulky. One...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether...

...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...and efficiently photoconverted in plant, Drosophila, and mammalian cells. Photoconverted GFP is compatible...Davidson MW, Looger LL. A bright and photostable photoconvertible fluorescent protein. Nat Methods. 2009...accidental discovery that laser treatment can photoconvert GFP from green to RED! This simple technique...time-consuming to repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery...discovery that GFP itself is photoconvertible, new studies of spatiotemporal dynamics can be undertaken using...

...emitted photons before they are destroyed. Factors that affect photostability The photobleaching rate of...will discuss how to select a photostable fluorescent protein. Photobleaching is the irreversible destruction...destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some point...have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display...likelihood that a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does ... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...to compare the photostability of different fluorescent proteins? Measuring photostability Since it is clear...

...illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly to the anionic form, causing...presented first, it causes irreversible photoconversion of the chromophore from the neutral to the anionic form...mEosFP, can form it’s chromophore at a more comfortable 37°C (5). Dendra2 photoconverts from green to red ...reversible PA-FPs (also known as photoswitchers) can be photoswitched from a dark state to a bright fluorescent... for vanishing, and pa, for photoactivation) (6). Dronpa is photoactivated by blue wavelengths of light...consider, such as brightness, photostability, pH stability, chromophore maturation rate at 37 °C, turnover...-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce though...

...biophysical interaction between two fluorophores. When the first fluorophore (the donor) is excited, it can...acids/lipids of interest are fluorophore labeled and that these fluorophores can undergo FRET when they ...targeting a FRET fluorophore to a biomolecule is a bit like a Fedex delivery. Your fluorophore package needs... energy directly via resonance to the second fluorophore (the acceptor) if the two probes are close together...physical distance between the donor/acceptor fluorophores (Stryer, 1978), a measurement that is unaffected...label your biomolecule of interest with FRET fluorophores? When is absolute labeling specificity needed...measure it). Molecular mailbox: How to get your fluorophore delivered to the right place? FRET experiments...

...technique! What is a photosensitizer? First, for a few definitions. A photosensitizer is a dye or protein... higher photostability than KillerRed, with KillerRed reaching greater than 75% photobleaching after just...system? Turns out that with light-inducible photosensitizers and a quick zap of the proper color light,...light, you can do just that.  Photosensitizing dyes and proteins have been around for awhile (check out this...Bruchez and Tsang labs recently developed a photosensitizer composed of the protein complex complex and... oxygen species (ROS). The ROS produced by photosensitizers inactivate target proteins or cells while ...having little effect on neighboring cells. The photosensitizer used in this work is composed of two parts:...

...called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore can emit...the photon budget and pushing the frontiers of fluorescence microscopy often requires more photons. For...cells can decrease the number of fluorophores, compromising the photon budget. Likewise, the switch from...desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...more photostable than fluorescent proteins, providing a straightforward way to improve the photon budget...rhodamine fluorophores, such as the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and... of photons for your imaging experiment. Innovative labeling strategies and improved fluorophores are ...

...Listen to the photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which...., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop...ratio, high sensitivity, responds quickly, is photostable, and is compatible with optogenetic control. ... Discovery of new protein binder tools for photoswitchable proteins cPYP and AsLOV2 Article contributed...temporally control cellular processes in vivo. Many photoactive proteins exist in nature, but developing them... either the light- and dark- states of the photoswitchable cPYP, which changes in conformation in response...bind to only the light- or dark- state of the photoswitchable proteins offers a powerful approach to control...

...wavelengths of emitted photons are higher than those of absorbed photons. When photons excite electrons of... you'll need to pick a fluorophore for each target and ensure the fluorophores, together, are able to ... about 380–700 nm. Photons of lower wavelengths are higher in energy, while photons of higher wavelengths...whereby a photon is emitted. This step is called emission. During excitation, some of the photon's energy...the energy of an emitted photon is lower than the energy of the absorbed photon. Correspondingly, wavelengths...outlining a few commonly used fluorophores and their emission spectra. Some fluorophores carry their emission ...and RB545. Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7...

...design utilized Magnet photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa...split fusions and generate a photoactivatable nickase and a photoactivatable repressor (dCas9). The activity...Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain...chemistry to photocage CRISPRs The aforementioned techniques each employed a photoactive strategy which...Comparison of photoactivatable CRISPR strategies Publication System nickname Photoactivatable moiety Cas9...et al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...optogenetic techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists...

...proteins predicted to be phosphorylated on at least one site. Phosphorylation is particularly important...predict. Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation rates than ...by FRET reporters having phosphorylated conformations that are less phosphatase-accessible. Kinase Translocation... signal (NES), both which are phosphorylatable. Prior to phosphorylation, the KTR localizes to the nucleus...biosensors Learn about using phosphoserine to study protein phosphorylation Additional Resources on Addgene.org...specific spectral properties that limit which fluorophores can be multiplexed together. Challenging  to...nucleus, but following phosphorylation bNLS activity is suppressed and NES activity is enhanced, leading...

...fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy is a fluorescence...decypher cellular dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists...worthwhile to test the photostability of your fluorescent protein when using 2-photon microscopy as this ... is advantageous when compared to photoactivable or photoconvertible proteins, as it enables tracking ...mobility with movement from the bone marrow and other lymphoid organs to specific tissues where they can scan... good tools to directly follow immune cells in lymphoid organs. The development of cell sorters together...several labs were able to decipher the journey of lymphocytes from the bone marrow to the thymus where they...

...brightness, photostability, and most importantly, a photoswitching ability. This photoswitching refers to...exciting the fluorophore at peak wavelength can also decrease the rate of photobleaching. Interaction ...function in live cells. Fluorophore properties: The specific properties of the fluorophore, such as excitation...fluorescence quantum yield, the ratio of photons absorbed over the photons emitted. For example, DAPI is much...Ghods, R. (n.d.). Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568-and Fluorescein...ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and fluorescent ligands. ...refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation...

...image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two drawbacks... leads to more rapid photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict...can lead to photobleaching when acquiring multiple images over a large axial range. 2-photon imaging, which...University. No matter whether you are a sports photographer at the Super Bowl, a medical technologist taking...background. In addition to this, the use of multiple fluorophores can add a second layer of contrast, color contrast...further exploit the unique characteristics of fluorophores to block out of focus fluorescence from reaching...large number of optical slices are acquired all fluorophores throughout the sample are constantly exposed...

...non-radiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of ...the two fluorophores used are in close vicinity. Thus, the distance between the two fluorophores and their...yield of the donor (number of emitted photons per absorbed photons) and the extinction coefficient of the... of a fluorophore pair, donor:acceptor stoichiometry, and cross-talk between the two fluorophore colours...microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple but limited...dipole-dipole coupling." The emission of the acceptor fluorophore can be measured using microscopy techniques. ...interactions within living cells. By coupling fluorophores to proteins, pioneers of this technique were...

...donor fluorophore to the acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the...absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor fluorophore--think of... of it as a virtual photon 3. Relaxation of the acceptor fluorophore back to its ground state by emission...acceptor fluorophores must be compatible, such that the emission spectrum of the donor fluorophore overlaps...emission of a photon with wavelength specific to the acceptor In order for FRET to occur several constraints...with the excitation spectrum of the acceptor fluorophore. Otherwise the energy transferred from the donor...

...Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon Generation Slower...emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus pyralis...Modification Not Required Required Photobleaching/Phototoxicity Not susceptible Susceptible Subcellular...accurate and safe. In the case of a luciferase assay, photon emission is measured resulting from the catalysis...luciferin, ATP and oxygen as substrates. Production of photons by this bioluminescent reporter occurs slower than... result of the different mechanisms to produce photons, chemiluminescent reporters are generally less ...background levels and improved signal sensitivity since photons are simply measured – they are not required to ...