... fluorescent HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules...created by Roger Tsien’s lab contain two fluorescent proteins (a FRET pair) separated by CaM and M13. When calcium...increases FRET transfer between the fluorescent proteins. FRET is highly amenable to biosensor creation... experimental setup? Consider what kind of instrumentation you have available, and the conditions of your... fluorescent proteins...

...its unbound state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore...flexibility is their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and...One of the advantages of oligonucleotides over proteins is their modularity, which is lost as soon as ...paved the way to new biosensor possibilities. For instance, further selection of Spinach with a target metabolite...applications that lend themselves to aptamer-based instead of protein-based fluorophores, including: Environments...Environments with higher temperatures, since proteins can denature while aptamers are more stable and can...Nucleotides, in general, are far less immunogenic than proteins. Assays that require recognition of ions or ...

... of toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active...that are insoluble, act on DNA, or are enzymatically active. For this reason, recombinant proteins are typically...most expression strains to accommodate high protein levels including:  ompT: Strains harboring this mutation...proteolysis of the expressed recombinant proteins. lon protease: Strains where this is completely deleted (designated...reduce proteolysis of the expressed proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction...Expression Systems Table 1: E. coli expression strains  Note: All strains are derived from the E. coli B strain...DE3) (RifR) Expression of unstable proteins Tuner (DE3)   Contains mutated lac permease whch allows for...

...strain. Common E. coli strains used in the lab Most of the commercial strains you find today are marketed...changes found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...glutamine insertion supF (tyrT)   Suppression of the amber (UAG) stop codon by tyrosine insertion λ-thi...of the popular strains, their genotypes, and their primary use in the lab. These strains are all based ...plasmids with R6Kγ origin; contains a mutant allele of the pir gene that maintains the plasmids at ~250 copies...optimal promoter for your gene of interest, painstakingly cloned into the perfect empty backbone, made...hard to count the number of different commercial strains of E. coli currently available – a quick Google...

...PUF proteins! A newly available PUF Assembly Kit makes it possible to devise RNA binding proteins to hit...applying the Golden Gate cloning method to human proteins known as Pumilio/fem-3 mRNA binding factors (PUF...step, researchers can now assemble designer PUF domains for RNA specificity engineering. “The RNA binding...University of Illinois at Urbana-Champaign. The PUF proteins include eight modular repeats, each of which binds...explained, the specificity of other RNA binding domains depends on the secondary structure of RNA. As a...eukaryotic RNA.” Endless possibilities with PUF proteins The tool she and her colleagues led by Zhao described...repressor. In principle, however, designer PUF proteins can be fused to other components to lend them ...

...career skills for positions both inside and outside academia. Inspiring future scientists At Addgene, we...

... at Albert Einstein College of Medicine. Several sets of near-infrared fluorescent proteins (NIR FPs) ...Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. ...Biliverdin chromophore Near-infrared fluorescent proteins (NIR FPs) were derived from natural photoreceptors...mouse. Image courtesy of Axel Nimmerjahn (Salk Institute for Biological Studies) Multiple uses: From...blue-light controlled optogenetics, such as channelrhodopsins and tools based on light-oxygen-voltage (LOV...Daria Shcherbakova is a faculty member at Albert Einstein College of Medicine. Her expertise is development...development and applications of fluorescent proteins, genetically encoded biosensors, and optogenetic tools....

...’s late father, Harold (Babe) Morgenstern. The origins of pBABE Well, it was way back in the mid 1980s...

...Even before fluorescent proteins (FPs) came into wide use, there were a variety of ways to monitor cell...cell, organelle, and protein localization. For instance, you might dye your cells and look at them under...hybridization experiments. In many cases fluorescent proteins have usurped old methods or complemented them ..., either in whole cells or as a pool of fusion proteins localized to a specific region of the cell. For...problematic aggregation-prone tetrameric fluorescent proteins can be. Cytotoxicity, improper localization, and...from previously developed monomeric fluorescent proteins mKO and mCherry, respectively. While Tsuboi et...blue-red maturation times (Table 1). These separate proteins should be useful for monitoring cellular events...

...about these proteins is that they are monomeric fluorescent proteins. Fluorescent proteins that oligomerize...University, Indiana University, and the Karolinska Institute developed mNeonGreen via 21 substitutions to LanYFP...fluorescent protein mNeonGreen. This fluorescent protein joins mTFP1 and mWasabi, as fluorophores from Allele Biotechnology...deposited at Addgene. What makes these fluorescent proteins unique and what can they be used for? Let’s take...Nathan Shaner and colleagues from The Scintillon Institute, Allele Biotechnology, Florida State University... sequence identity to other common fluorescent proteins and therefore can be used in immunoprecipitation...immunoprecipitation experiments with other fluorescent proteins. Find mNeonGreen plasmids here! mTFP1: a monomeric teal...

...fluorescent proteins requires some thought. The first step in choosing fluorescent proteins for your multi-color...aware of what fluorescent proteins are available. With new fluorescent proteins being published every month...fluorescent proteins, I maintain an interactive graph and table of the best fluorescent proteins currently...Choosing compatible fluorescent proteins To choose a set of fluorescent proteins to be imaged together, you...between the two fluorescent proteins. If you must image fluorescent proteins whose spectra overlap, there...fluorescent proteins, but these are beyond the scope of this post. Are your fluorescent proteins compatible...wavelength red fluorescent proteins like TagRFP or mRuby2 than longer wavelength proteins like mCherry. For background...

...fluorescent proteins found on the red side of the spectrum are not GFP derivatives, but are instead related... of use, GFP and other fluorescent proteins have become a mainstay in molecular biology. Scientists can...study the interactions between two proteins or between two domains of a protein that undergoes conformational...Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed...aqueous solvent. This tightly-packed structure explains the importance of the entire GFP protein, which.... Table 1 below lists a few common fluorescent proteins and their mutations relative to wildtype GFP. ...red-fluorescent protein repertoire; however, these proteins are unique from GFP and the mutation definitions...

...Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses. Feng S et ...measure the signal produced when the candidate proteins’ interaction brings the pieces of the FP back ... to help!  General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments...canonical beta-barrel structure. Most frequently, the proteins are split between strand 10 and 11 to yield large... with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R,...(1-10) and mNG2(11) Improved split fluorescent proteins for endogenous protein labeling. Feng et al. Nat...

...deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one...conductance compared to current channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain...engineering channelrhodopsins There are three main challenges to engineering channelrhodopsins to have improved...membrane localization of transmembrane proteins, like rhodopsins, are highly constrained and poorly understood...understood. It’s hard to design new channelrhodopsins with high-throughput proteins engineering methods, like directed... than existing channelrhodopsins CoChR and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic...expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive. When light...

...which other proteins it forms complexes with. These “colocalization” studies give insights about the proximity...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...its role in a disease, researchers often isolate proteins of interest and examine them using biochemical...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...You can take advantage of the many Fluorescent Proteins (FP) available for microscopy, by cloning your...final destination. As an example, most secreted proteins are produced in the endoplasmid reticulum, modified...extracellular destination. At the end of their life cycle, proteins may be transported through the vesicles to the...

...binding proteins waiting to be explored and exploited. With better characterization, these proteins could... sequence the inserted DNA and characterize your new putative host cell-binding proteins! Many thanks...their bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages)...constitute one of the most popular classes of phage proteins to study. These are highly useful for the biotech...Addgene's MIcrobiology Pages The problem: Host binding proteins are hard to find Sequencing and cloning phage ...that allows for a phage genome to be probed for proteins with host-binding activity without the need for...genome that encode for functional cell-binding proteins in the (all-too-common) event that homology-based...

...from Joachim Goedhart: Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection.  mEmerald...Updated May 10, 2021. In the world of fluorescent proteins and their use for imaging cell biology, Michael...impressive 1,350 scientists from more than 275 institutions all over the world. In the course of all those...plasmid backbones spanning a rainbow of fluorescent proteins “It’s a great collection with over 300 backbones...interest isn’t in the ORF collection, the fluorescent proteins are, which makes mixing and matching a relatively...exponentially as far as new targets and fluorescent proteins are concerned.” For more information on choosing...targets since the Davidson lab always tagged new proteins with mEmerald first, Baird says. From F-Actin ...

...immunohistochemistry methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins to facilitate its detection... that fluorescent proteins might not always be the best option. Fluorescent proteins can interfere with... on the Addgene blog: Fluorescent Proteins 101: GFP Fusion Proteins – Making the Right Connection.   ...activity of neurons and other brain cells. Since the opsins or chemogenetic receptors used in these techniques...detection. Fluorescent proteins are most widely used tags because they emit endogenous fluorescence that... vivo, or post-mortem experiments. Fluorescent proteins can also be identified using antibodies and immunohistochemistry...immunohistochemistry techniques. Thus, these proteins provide a versatile method to identify the expression...

...Addgenie Eric Perkins presented a poster a few years ago at a CRISPR meeting.Read his Instagram feature here... what lies ahead. Thus the “Meet an Addgenie” Instagram interview series was born. Over the six months... Addgene’s Director of Product Management Eric Perkins, who has been in many roles in Addgene since he... really get me where I haven’t gotten myself.” Perkins noted that there is a more emphasis today about...Shannon Rinaldi in the Addgene lab! Read their Instagram profiles here (Kenkel, Rinaldi)! But leaving...Vedantam with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya...Read all of the Meet an Addgenies features on Instagram! Staying true to your values While going through...