...latest protocol video, "Coomassie Purity Stain of Recombinant Antibodies."  Hot tip! This protocol can easily...helpful, you can find our complete collection of protocol videos on the Addgene YouTube page. ...

...in E. coli easier and more robust. Synthetic photobiology: why use light? In synthetic biology and bioengineering...two-component system (TCS). Levskaya et al. fused a photosensory domain from cyanobacteria to a common E. coli...Tabor et al. subsequently engineered a second photosensitive TCS activated by green light; these two systems...two-component system (TCS). A hybrid histidine kinase/photosensor senses various light inputs, and an obligate ...

...can check out all of our protocols on our Protocol page, and find our protocol videos on our YouTube page...for first-timers. That's why we gave our written protocol a video companion! Follow along as Addgenie Meghan...

...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... 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... less similarity. For these three kinases, photoswitchable Dronpa domains were inserted at sites homologous...locations they were attached to in MEK. The photoswitchable versions of MEK1, MEK2, Raf1, and CDK5 all ...

...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..., a photoconversion signal may not be observed. Sattarzadeh et al. found that only the protonated form...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...

...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...have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display... 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... to quantify the photostability of fluorescent proteins. To determine photostability, an experiment is...Measuring photostability at realistic powers will provide better insight into the photostability in the ...

... a mixed population of neutral (protonated) and anionic (deprotonated) forms of the chromophore which ...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...your activation protocol for your particular set up. Applications of photoactivatable fluorescent proteins...-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce though...containing inactive PA-FPs is photoactivated, imaged, and then photobleached. Additional PA-FP molecules...do not emit the large number of photons achievable with photoswitchable dyes. Correlative light and electron...

...materials and information,” we love to share lab protocols that we use as we serve the scientific community...community. Here are a few virus-related protocol videos that may help to save you time and improve your experiment... purification.  Check out the AAV Purification protocol page for more detailed instructions. AAV titration...curve, visit the AAV Titration by qPCR written protocol page. Getting started with tissue culture The...tissue cultures. To keep up on all of the latest protocol videos, lab tips, and more video content from ...video series Resources on Addgene.org Find lab protocols on the Addgene website Watch Addgene's video collection...

...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:...

...finally characterization. Read and download the full protocol from the JOVE article here on Addgene's website...

... remote learning due to COVID-19. We had many protocols for plasmid cloning and working with viral vectors...learn the ropes! All material can be found on our Protocols page and YouTube page.  Happy learning, and welcome...

...started on our deposit page. Protocols.io Website: http://protocols.io/ Protocols.io is the creation of ZappyLabs...wiki/Protocols/Template). Nature Protocol Exchange Website: http://www.nature.com/protocolexchange/ Nature...biomedical topics. Protocol Online Website: http://www.protocol-online.org/ Protocol Online is one of the...the new protocols.io beta platform for sharing, annotating, and storing life science protocols. Since ...popularity, I decided this protocol would be a worthy first contribution to the protocols.io community. Did I mention... updated protocols So that’s great, but what if someone in China improved upon that protocol? How would...comment, annotate, and edit protocols. After my introduction to protocols.io, I decided to research what...

...encoded fluorescent sensor for serotonin. According to Unger, “serotonin is a well-known but poorly studied...the sensor for serotonin by over four orders of magnitude. Unger validated the serotonin sensor with proof-of-principle...firing of serotonergic neurons (measured using a red calcium sensor) leads to the release of serotonin (measured...use the serotonin sensor in sleep-related studies and in assays for measuring human serotonin transporter...Chiasson explains. Elizabeth Unger: Developing a serotonin sensor The goal of Elizabeth Unger’s postdoctoral...there are no known bacterial proteins that bind serotonin. Unger redesigned the binding pocket of the periplasmic...periplasmic binding protein so that it would recognize serotonin. Using a combination of computer modeling, directed...

...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...

...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...Otabe T, Hasegawa S, Yoshimoto K, Sato M (2017) CRISPR–Cas9-based photoactivatable transcription systems...

...amplification protocol developed for each individual pooled library; generalized amplification protocols tend ...reference protocols There are many decisions to make when developing your amplification protocol! We recommend...one-size-fits-all” protocol! Each library is different, and you should always experimentally test the protocol you design...surprised to learn that we ask for an amplification protocol with your deposit. This is because repeated amplifications... we’ll discuss how to develop an amplification protocol for your pooled library deposit. When a requesting...consider when creating a pooled library amplification protocol. Created with BioRender.com.   The most important...efficiency you need to achieve. Most pooled library protocols aim for somewhere between 30x and 200x coverage...

...fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy is a fluorescence...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 ... about one millimeter in depth. Two low-energy photons (typically from the same laser) cooperate to cause... right). The excitation generated by these two-photons occurs only at a chosen focal volume and thus the...specimens. See the video below for an example of two-photon intravital microscopy from PLoS Pathogens - Kamenyeva...clear view of cellular movement in real time. Two-photon microscopy was the next revolution in cell imaging...

...specific protocol for producing virus, I recommend that you follow that protocol. If not, our protocol on Lentivirus...the protocols provided by the depositing lab whenever available. Addgene also shares the protocols we’...these are not general amplification protocols. Rather, each protocol is optimized for a single, specific...amplification protocol, or have constructed your own library, you’ll need to develop your own protocol that amplifies...lentiviral CRISPR libraries. Protocols from the Broad Institute- A variety of protocols and guides regarding ...with a colony formation titering assay or similar protocol. After functional titer is determined, larger ...  Figure 2: A pooled library page with protocols boxed in red.    Unlike a standard single ...

...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...approximately half the energy of blue light, two photons of red light are needed to excite GFP compared ...the objective can a a high enough density of red-photons be establish for this to happen.Using red light...be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues. Light...

...the photon budget and pushing the frontiers of fluorescence microscopy often requires more photons. For...more photostable than fluorescent proteins, providing a straightforward way to improve the photon budget..., higher fluorogenicity, photoactivation etc.—can further increase the photon budget and allow us to push...small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances lead to an ... chemists? One reason is something called “the photon budget”: each sample has a limited number of fluorophores... each fluorophore can emit a limited number of photons before bleaching. The amount of information one...decrease the number of fluorophores, compromising the photon budget. Likewise, the switch from ensemble imaging...