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...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-...High-throughput screening strategies Although the tools described above make CRISPRa more accessible to neuroscientists...The pooled nature of this experiment requires fluorescence activated cell sorting (FACS) to isolate differentiated... in vitro and in vivo applications, the tools described above make CRISPRa in the brain relatively easy...

...sample. A variety of chromogenic, chemifluorescent, and chemiluminescent substrates are available. Labs ...standard plate reader as opposed to chemifluorescent, and chemiluminescent methods that require specialized...peroxidase (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we ...

...successfully and expressing the protein. For example, fluorescence microscopy images resulting from our experiment...was not successful due to the absence of GFP fluorescence from the living, viable cells. This result presents...positive control, the internal control vector, was described earlier. Once you are sure your conditions are...produces the expected effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid...come from multiple, independent sources. We'll describe these a bit more below, but the papers cited in...

...several secondary antibodies, increasing the fluorescence signal intensity. However, you need to be careful...sorting based on a single (lineage) marker, while fluorescence-activated cell sorting (FACS) enables you to...off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant. With each cell division...(Figure 4a), yielding a stepwise decrease in fluorescence intensity in a (histogram) flow plot (Figure...division half of the dye molecules are lost and the fluorescence signal weakens. (b) The heterogenicity of proliferative...

...site-specific incorporation of phosphoserine (as described below) provides researchers with a valuable tool...context-specific effects of UAG suppression, as described (6). To determine the stoichiometry of phospho-incorporated...phospho:non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid...-Sung, et al. "Expanding the genetic code of Escherichia coli with phosphoserine." Science 333.6046 (2011...al. "A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation...

...of working with fruit flies is their ability to escape their habits and live in the lab itself. Many of...Light Interval System, or FLIS, incorporates a bioluminescence protein into the chitin that can be activated...everywhere find and re-contain any specimens that escape during routine lab activities.  Here, we demonstrate...plasmid-based system for locating and containing escaped flies. Nature 4:1-22. https://doi.org/4.01.2022...

...process of mouse model generation simpler, as described in an earlier post on generating CRISPR mouse ... make. For example, large insertions, like a fluorescent protein knock-in, suffer from low efficiency ...with your desired insertion feature (e.g., a fluorescent protein), you can amplify the desired region ...most of the commonly used cassettes, such as fluorescent proteins (EGFP, mCherry etc.), recombinases (...'d use ivTRT to create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids...

...delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral vector background Until ...lentivirus-derived vectors are capable of transducing certain quiescent or terminally differentiated cells such as macrophages...interest is expressed together with a marker (Fluorescent protein or selection marker) allowing for selection...process is to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the...quantify viral RNA. All of these methods have been described in a previous Addgene blog Once you know your ...

...Specific C-to-U Exchange, or RESCUE. Read the paper in Science Find RESCUE plasmids If you have a new... toolkit for targeted genomic integration of fluorescent tags in neurons. Their toolkit named ORANGE (...Editing) mediates targeted genomic integration of fluorescent tags in neurons.  Read the paper in bioRxiv ...

...(FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...via applications such as western blot and immunofluorescence. GST  Glutathione S-Transferase (GST) is ...

...camelids too, though not just for the reasons the UN describes. In honor of the Camelid family, I wanted to take...of times on this blog over the years and have described how they compare to other antibodies and affinity...single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens...sdAbs in diagnostic applications. For example, fluorescently and radioactively labeled sdAbs have become ...

...database describing nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all ...authors of the paper in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by...Cellosaurus received a report that HT-55, which was described as a colon cancer cell line, is actually a rectal...

...mCherry fluorescent protein followed by a stop codon and the coding sequence for a green fluorescent protein...representation of cell-free system, capable of producing fluorescent proteins and enzymes. Image from Moore et al....validated through monitoring the expression of fluorescent proteins and oxytetracycline enzymes. Next, this...

...used for western blot, flow cytometry, and immunofluorescence . However, protein purification is not ideal...for immunoprecipitation, western blot, and immunofluorescence with some success for protein purification...and can serve as an epitope tag as well as a fluorescent marker. The GFP tag is ideal for live cell imaging...imaging, FRET, flow cytometry, or immunofluorescence where an antibody can’t be used or that step can ...

...recent contribution to the field is a series of fluorescence resonance energy transfer (FRET)-based calcium...with a solution: they devised self-associating fluorescent domains whose association is disrupted in the...Robust red FRET sensors using self-associating fluorescent domains. ACS Chem Biol. 2013 Oct 18;8(10):2133...et al. MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009...

...see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity... cells were labelled with a green or violet, fluorescent dye, respectively. WT and KO cells were mixed...through an epitope tag such as FLAG/His/GFP. Fluorescent protein tags, which correlate closely with target...recognized by either antibody was not shared. Tables describe that the antibodies both recognized the target...use in western blot, immunoprecipitation, immunofluorescence and flow cytometry. F1000Research, 13, 817...

...Choose the Brightest Fluorescent Protein Resources on Addgene.org Find Fluorescent Protein Plasmids Check...mRNA this displacement leads to a change in the fluorescent signal: untranslated RNAs appear yellow, and ..., which contains the tethering sites for the fluorescent proteins and is driven by an inducible promoter...

...speed of light over the last decade, but the fluorescent proteins (FPs) necessary for many of these applications...light and electron microscopy and observed high fluorescence resilience to these preparations. After crystalizing...    Fig. 4: hfYFP retains folding and fluorescence in high molarity GdnHCl, at high temperatures...!   Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy. Nat Methods...

...Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed...

...containing two fluorescent proteins and four repeats of a miRNA target site. One fluorescent protein (EBFP2...location and function of microbes rely heavily on fluorescent protein genes. These optical reporters have limited...successfully visualized in vivo using ultrasound in Escherichia coli and Salmonella Typhimurium. This work demonstrates...