... disrupted when GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the ...heterotrimeric G-alpha subunit fusions that do not tolerate N- and C-terminal fusions (Adjobo-Hermans, 2011)....microscope, and finally (iv) good toleration in fusion proteins. Since GFP is such a well-validated, all-round...interfere with the activity or the localization of the fusion protein. Also, in cases where the protein of interest...interest is relatively small, adding GFP may alter diffusion rates. Smaller genetically encoded probes are .... GFP can only be used for tagging proteins GFP fusion proteins are made by connecting its DNA code to...are defined by a gRNA. Since Cas9 is a protein, a fusion with GFP allows for detection of genomic locations...

...interest fused to the C-terminus of SCFP3A Amber N1 Yellow Mammalian Express a gene of interest fused to ...interest fused to the C-terminus of Amber mVenus N1 Yellow Mammalian Express a gene of interest fused to ...can be used to create your choice of fluorescent fusion protein with your gene of interest: Plasmid ...mCerulean N1 Cyan Mammalian Express a gene of interest fused to the N-terminus of monomeric Cerulean mCerulean...mCerulean C1 Cyan Mammalian Express a gene of interest fused to the C-terminus of monomeric Cerulean mTurquoise2... C1 Yellow Mammalian Express a gene of interest fused to the C-terminus of monomeric Venus pCEP4YPet-MAMM...SYFP2 Yellow Mammalian Express a gene of interest fused to the C-terminus of SYFP2 Clover Green Mammalian...

...orientation of the BirA* fusion matters! Schopp et al saw different levels of fusion protein expression and...BirA, an E. coli biotin ligase. In BioID, BirA* is fused to a bait protein and it biotinylates proteins that...Split-BioID method, each BirA* fragment is then fused to one of two bait proteins that are known to interact...proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed in cells via plasmids or...C-terminal BiraA* (C-BirA*). These two fragments are fused to two proteins, here proteins Y and Z, that are..., and that result in proper localization of the fusion protein. Advantages of Split-BioID Split-BioID’... remains even if the interaction with the BirA* fused bait proteins ceases. Lastly, Split-BioID and BioID...

...are described in more detail below: Fusion protein construction Fusing FP fragments to your PIPs requires...determine where to fuse the FP fragments. In the absence of this information, you can fuse each FP fragment...constructing fusion proteins, check out our article: Fluorescent Proteins 101: GFP Fusion Proteins - Making...hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity and present in large...fluorescence. When complementary FP fragments are fused to two hypothesized protein interaction partners...more difficult with FRET, as the overexpression of fusion proteins it requires can lead to aberrant subcellular...BiFC experiment generally involves three steps: fusion protein construction, cellular expression, and ...

...Fluorescent protein fusions One of the easiest ways to track a given protein is to fuse it to a fluorescent...Instead of directly fusing a fluorescent protein to your protein of interest, you instead fuse it to the synthetic...any case you would use a traditional FP fusion, a SunTag fusion could be used as well. Pros of SunTag ...system is far from perfect. For example, If your fusion protein is expressed at low levels, you have to...copies of the short epitope GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed... 24 kDa for GFP alone. Although traditional FP fusions can also affect protein activity, half-life, or...

...dCas9-KRAB fusion, both targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64...Cas9 for your knockout experiment Generating a no-fuss genetic knockout  Knock-out cells or animals are... editors are dead Cas9 (dCas9) or nCas9 enzymes fused to either cytosine or adenosine deaminases to facilitate...various cargo to a specific DNA locus by directly fusing the cargo to dCas9.  This technique has been exploited...transcriptional repressor domain which is frequently fused to Cas9 for this purpose. dCas9-KRAB plasmids for...simplest of the dCas9 activators consists of dCas9 fused to a single transcriptional activation domain - ... an epitope tagged dCas9 and antibody-activator fusion proteins. The Synergistic Activation Mediator complex...

... three elements. Broadly: A Cas nickase or Cas fused to a deaminase that makes the edit A gRNA targeting...the inactive dCas9 (Komor et al., 2016). These fusions convert cytosine to uracil without cutting DNA....converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase (UGI) to...editor using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida et al., 2016). Target-AID...minimum efficiency of ~50% and confirmed that the fusion protein retained the PAM properties of the individual...further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to the N-terminus of BE4...editors into one? This has recently been done by fusing the adenine and cytosine editing components together...

... same protein of interest tagged with different fusion proteins for different experiments over the years...idea. What if there were a way to just make one fusion protein that switches to any color or tag you want...molecule you choose (Figure 1). Just express the fusion protein and add a specific ligand! A self-labeling...structure is the protein of interest, and black is the fusion tag or labeling system. Image adapted from Lavis...2003, Kai Johnsson’s lab developed a self-labeling fusion tag based on the human DNA repair protein AGT that... Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR) could...the tip. 649-mDia1 and 549-CP are both SNAP-tag fusions, purified and labeled separately with different...

...transcriptional activators: some methods rely on fusion proteins while others re-engineer components of...Description CRISPR activation can occur through fusing dCas9 with VP64, a strong transcriptional activation...transcription. This is done through creating a dCas9/VP64 fusion protein engineered with aptamers that bind to MS2...dCas9, SunTag uses a repeating peptide array to fused with multiple copies of VP64. By having multiple...consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to activate transcription...notable CRISPR activators is that it requires a fusion protein, rather than relying on a two-component... activation. dCas9-CBP In this method, dCas9 is fused to CBP, an histone acetyltransferase domain capable...

...ternary complex formation between the FKBP12F36V-fusion and the ubiquitin proteasome machinery through ...expression of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific knock-in of FKBP12F36V...running in your lab Confirm the functionality of your fusion chimeras For each protein-of-interest, it is important...of comparing functionality of N- and C-terminal fusions and degradation in a cell line of interest prior... used to evaluate degradation of the FKBP12F36V-fusion chimera of interest. In the cell lines we have ...provide the best confirmation of selective FKBP12F36V fusion chimera degradation. In the cell lines we have ..., Erb et al., Weintraub et al.), assessments of fusion chimera functionality (Nabet et al., Erb et al....

...by splitting Cre and fusing each half with PhyB or PIF6, phytochrome-like fusion proteins from the plant...unmodified enzyme. Each half of the base editor is fused to a fast-splicing split intein and co-infected ...plasmids containing a red-shifted Ace-mScarlet, a fusion of a voltage-sensitive inhibitory rhodopsin (Ace2N...mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive inhibitory rhodopsin...soon as red light or PCB is no longer present, the fusion proteins separate, inactivating Cre. This is critical...Cre (CreC) and the N-terminus of Cre (CreN) are fused to the truncated Phytochrome B (PhyBΔ) and the active...

...reversible oligomerization when fused to a protein of interest. Fusion protein oligomerization happens...cryptochrome-interacting basic-helix-loop-helix) protein. After fusing the CRY2 and CIB1 domains to separate proteins ...used to control homo-oligomerization of a protein fused to CRY2, although some studies have shown that this...derivative of the opsin CoChR, termied soCoChR, by fusing a fragment of the kainate receptor subunit to the... the C-terminus of the protein. Unlike the diffuse neuronal expression observed with wild type CoChR, ...which TagRFP-T and Dronpa flanked a PKA substrate fused to the phosphopeptide binding domain FHA. The FHA1...

...repressor) fusion protein, the authors show that the VP16 transcription activation domain fusion increases... in cellular functional imaging and in protein fusions. The Gadella lab has deposited all mScarlet constructs...activity. In addition, proteins can be “caged” by fusion to specific domains thereby rendering them inactive... base editing technique uses dCas9/Cas9 nickase fused to a cytidine deaminase for targeted conversion ...minimum efficiency of ~50% and confirmed that the fusion protein retained the PAM properties of the individual...the B. subtilis libraries Transcription factor fusions elucidate iPSC reprogramming mechanism The Ptashne... efficiency of iPSC reprogramming while the HP1 fusion eliminates reprogramming. These results argue that...

...validate that the fusion protein localizes properly by comparing the signal from the fusion itself to the ...fluorescent tag. Once expressed as a fluorescent protein fusion, it is possible to track your protein of interest...finally degraded (2, 3). Once you've created your fusion protein, it's important to validate its function...always possible, to use inert fluorescent protein fusions to visualize subcellular structures. For example...example, you might use your fluoresent protein fused to a signal sequence that traffics the fluorescent protein...size – it is possible that a large or charged FP fusion can change the subcellular localization of a protein...

...are just one (admittedly large) class of common fusion protein tags. Biochemists and molecular biologists...amassed a considerable molecular tool box of tags and fusion proteins to aid in the expression and purification..., glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purification...immunoprecipitating, or purifying that protein may be to fuse an affinity tag to it. The FLAG, hemaglutinin antigen...column. Like other affinity tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike... available, and though adding too many tags and fusion proteins to your protein of interest would eventually...scientists use tags, with a few time-tested tags and fusion proteins as examples. The tables list more common...

...vacuolar/lysosomal and cytoplasmic Rosella. When fused with a mitochondria-targeting sequence, Rosella ...mitochondria, or mitophagy. Rosella can also be fused to a protein of interest to track the autophagy ... This autophagy biosensor contains mRFP and GFP fused to the autophagy-related microtubule-associated ...puncta turning red (GFP negative/RFP positive) upon fusion of an autophagosome with a lysosome. An increase...to more yellow and red puncta, while a block at fusion of autophagosomes with lysosomes leads to an increase...Fluorescent Protein, but following translation, this fusion protein is cleaved to generate one molecule of ...

...Nano-lanterns contain a Renilla luciferase variant fused to an FP; when supplied with a luciferase substrate...applications, including optogenetics, biosensors, and fusion proteins. Solving the problems of low luminescence...Saito et al. described the first Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with...Renilla luciferase variant are used to excite the fused fluorescent protein. This technique is very similar...compartments over several minutes using Nano-lantern fusion proteins. To further improve long-term imaging,... as good reporter genes for endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent...

...); and Casilio modules (fusion proteins consisting of effector domain(s) fused to PUF domains programmed... programmable DNA binding protein. dCas9 can be fused to different effector proteins to mediate functions..., in most cases, effector proteins are directly fused to dCas9. CRISPR-Cas9 combinatorial functionality...hybrid construction of CRISPR-dCas9 and effectors fused to the programmable Pumilio (PUF) RNA-binding domain...acetyltransferase modules, cloning vectors for PUF fusions, and more (see table 1). Table 1: Casilio Modules...

...turnover rates. Additionally, by engineering FP fusions associated with cellular organelles, scientists...processes, including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although... pitfalls of overexpressed fluorescent protein fusions is to replace the genomic copy of your gene of ...of interest with a fluorescent protein fusion. CRISPR genome editing can help accomplish this goal. The..., allows users to easily create C-terminal tag fusions of endogenous genes in human cells. To use CRISPaint...users to generate a plasmid containing a direct fusion of the desired gRNA to the U6 promoter, and a second...

...Monitor dynamics of vesicle fusion and release from the plasma membrane by fusing various vesicle cargoes ...more popular timers (whether as part of protein fusions or expressed independently) originally fluoresce...expressed, either in whole cells or as a pool of fusion proteins localized to a specific region of the ...expressing a single FP-timer protein or protein fusion. This one protein method simplifies cloning and...between models for trafficking to the lysosome by fusing Medium-FT to LAMP-2A, a lysosome-associated membrane...