...costly to share. What is a RANbody? A RANbody is a fusion between a reporter and a nanobody that’s encoded...J.R., & Yamagata, M. (2018). Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical...

.... Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on a reduced glutathione...glutathione matrix. The advantage of a fusion protein is the efficient purification of an already immobilized...for kinetic analysis Proteolytic removal of the fusion protein could provide a more relevant kinetic analysis...produces biotin ligase and a protein of interest fused to maltose binding protein (MBP, New England BioLabs...protein in bacteria and subsequently purify the fusion protein from a bacterial extract. The MBP tag can...using Xa protease. The carboxyl-terminus of the fusion protein includes an AviTag and a 6X HisTag for ...protein we tested, it may be possible with other fusion proteins - but it would remove both the AviTag ...

...Fluorescent Protein Fusions Observing a bright signal for the first time after cloning an FP fusion with your favorite...consequences of misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains...misfolding impacts fusion protein function and this could lead to under and overestimation of fusion protein activity...impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology...interest is exciting and rewarding. But, what if an FP fusion with a resident endoplasmic reticulum (ER) protein...troubling, what if a significant fraction of the fusion protein molecules fail to correctly fold? Misfolded...localization and dynamics of fluorescent protein fusions in live cells in real time. Previously, we have...

...engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping...Creating fusions at the N- and C- termini of your protein of interest The most straightforward fusion protein...use terminology like “C-terminal fusion,” but I find this very confusing because it can mean that the fluorescent...cloning site for generating fusions. For the POI-fluorescent protein fusions, our experience is that a ...For the fluorescent protein-POI fusions, we generate straight fusions, without any linker. When both orientations...example, the APT1-mVenus fusion localizes at the Golgi, whereas the mVenus-APT1 fusion does not (figure 2)....fluorescent protein fusions There are several choices to consider before generating a fusion protein, e.g. ...

... them to a tumor - they used focused ultrasound (FUS) to heat the tumor area, as the image below shows...stable levels for 6 days - after just 15 minutes of FUS.  Similar constructs to the one above can be made...

...events that occur in less that 5% of the population (Fu et al. 2013, Vouillot et al. 2015). Meanwhile, Sanger...sequence requirement at the terminal 3’ PAM site (Fu et al. 2013; Pattanayak et al. 2013). This approach...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...

...guides (19-20bp)?  Small-scale studies (Tsai et al., Fu et al.) suggest 17-19 bp long guides have reduced...PMID: 25513782. Pubmed Central PMCID: PMC4320685. 3. Fu, Yanfang, et al. "Improving CRISPR-Cas nuclease specificity...

...PMID: 26829887. PubMed Central PMCID: PMC4735761. 6. Fu, Y. et al. High-frequency off-target mutagenesis ...

...10.1016/j.neuron.2010.11.021  Gambarotto D, Zwettler FU, Guennec ML, et al (2019) Imaging cellular ultrastructures...

...him on Twitter.   References Arribere JA, Bell RT, Fu BXH, Artiles KL, Hartman PS, Fire AZ (2014) Efficient...

...Viral delivery methods include focused ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV...widely throughout the organism to target large or diffuse biological structures. Of primary interest here...Stanton, A. C., Lagerborg, K. A., Tellez, L., Krunnfusz, A., King, E. M., Ye, S., Solomon, I. H., Tabebordbar...Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J. J., Messemer, K...

...gene activation when fused to dCas9. In the dCas9-SunTag-VP64 system, dCas9 is fused to a scaffold containing...hook proteins fused to a streptavidin core that are able to retain reporter proteins fused to the streptavidin-binding...interest is fused to an sgRNA used to direct Cas9 to a specific genomic locus. When fused to the sgRNA...fluorescent protein fusions that can be used for monitoring cellular dynamics. These fusion proteins have been...When fused to an RNA of interest, Broccoli-DHFBI fluorescence can be used to visualize the fusion in vitro...or activation, the c-terminal cas9 fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS...light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein domains CibN and Cry2...

...single fusion protein, as in a FRET biosensor, then the local concentration of FPs around that fusion protein... can lead to polymerization of the fusion protein. A dimeric fusion protein will have two FP binding domains...polymer formation as fusion protein concentration increases. The polymerized fusion proteins that form ... blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile..., there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the...noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could...tetramers (i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs ...

...of interest(POI’s) are fused to TAs and DBDs (see Figure 2a) (4). These fusions are then co-expressed ....g. transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants...period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers...effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown is still able to bind to the upstream...containing the target sequence of the DBD. The DBD-POI fusion (commonly referred to as the “bait”, Figure 2b)... two POI’s interact, this will bring the POI-TA fusion (commonly referred to as the “prey”) in close enough... reconstitutes functional luciferase only when fusions of the two halves of the luciferase protein interact...

... of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1;...CIB1 and it was fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length...can also be achieved using single-chain fusion proteins by fusing Cas9 to photoreceptors. One prime example...- the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65...p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by...the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below. Once...tested a variety of combinations to optimize both fusion proteins. The best performing combination was NLS-dCas9...

...tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol Biol Cell...molecular biologists to fuse fragments of DNA together in a desired order and express fusion proteins in a target...SpyCatcher fusion that can be anchored to amylose resin for solid-phase synthesis of larger fusions through...to generate mAID fusions to endogenous genes. Cell lines with the appropriate fusions could then be selected...problems by fusing an RBP to the catalytic domain of RNA editing enzyme, ADAR. This RBP-ADARcd fusion protein... to easily create protein expression constructs fused with epitopes or fluorophores, generate expression...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with the catalytic...

...epigenome-engineering tools - zinc finger nucleases and TALENs fused to epigenetic modifiers enabled epigenetic modifications...Researchers showed that TALE-TET1 constructs, which fused a TALEN to the Tet1 demethylase catalytic domain...various promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate...targeting much easier! With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier...CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic modifiers to specific...Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase...has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from ...

...proteins can interfere with fused protein function Under some circumstances, the fused fluorescent proteins ...immunohistochemistry methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins to facilitate its detection...could affect the properties or trafficking of the fusion protein (Kimple et al., 2013). We found that the...initially used a construct in which the hM4Di was fused to mCherry. To investigate the localization of the...neurons, we next hypothesized that a different tag fused to the hM4Di might improve the transport of this...the influenza virus, introduced to the field of fusion proteins more than thirty years ago (Field et al...also found that this is not generalizable to all fusion proteins. For example, we saw that another type...

... NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc fused to the...produce a series of FP-RLuc8 fusions for multicolor imaging. In all of these fusions coelenterazine intiates...plasmid #85203) NLuc fusions. The Nagai lab has additionally deposited more than 25 fusions of the Enhanced... Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes...better biosensors. NanoLuc®-fluorescent protein fusions for imaging Fluorescent proteins are fantastic ...the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited... by Nluc emission and the lab has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs...

... 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...