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...switched on and off reversibly. This ability to modulate fluorescence allows scientists to observe single... (T203H) (1). wtGFP normally contains a mixed population of neutral (protonated) and anionic (deprotonated...T203H mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display...with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs by performing several...photons achievable with photoswitchable dyes. Correlative light and electron microscopy (CLEM) is an imaging...Ryoko, Hideaki Mizuno, and Atsushi Miyawaki. "Regulated fast nucleocytoplasmic shuttling observed by reversible...

...site-specific recombinases, regulatory elements, developmental regulators, and lots of viral components... as Pichia pastoris). If your goal is protein isolation, then the MoClo Pichia Kit will get you started...developed to study the basic biology of and gene regulation in the opportunistic pathogen Acinetobacter baumannii... Molecular Tools to Study Gene Expression and Regulation in Acinetobacter Baumannii.” ACS Synthetic Biology.... “A Unified Multi-Kingdom Golden Gate Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer ...

...vector production, assembled AAV vector particles accumulate inside the producer cells and purification protocols...gradients. The current purification protocols for isolating exo-AAVs rely on differential centrifugation steps...Figure 2). Since the development of the exo-AAV isolation protocol by Maguire and colleagues, other researchers...et al., 2017), and exo-AAV8 expressing human coagulation factor IX in liver in a mouse model of Haemophilia...et al. 2018). The structure of exosome AAVs is related to the use of microvesicles as a vehicle for drug...

...1, to create a “cumulative wealth distribution” curve, or Lorenz curve. By calculating the area under ...can be counteracted by growing the cells on solid plates, where each transformant forms its own colony, ...less of a concern, but Addgene still recommends plating the transformation mixture on solid media. The ...The colonies are then scraped off the plates and pelleted for DNA extraction. Prepare library product for...

...Antibodies, 1999). This creates an immortal cell population that will continue to produce the specific antibody...or, are injected with the whole protein. After isolating the B cells from an immunized animal, those B ...step. Then, a limiting dilution is performed to isolate individual hybridomas and ELISA is used to identify...the selected hybridoma is expanded as a clonal population (Holzlöhner et al., 2017). Why choose monoclonal... Addgene’s antibody plasmid collection Read the latest on Addgene's new antibody project ...

...gamma-retrovirus is Murine Leukemia virus (MLV). Naturally isolated gamma-retroviruses have been associated with cancer...lentivirus and gamma-retrovirus are so closely related, their required components are very similar. While...heterologous envelope or a post-transcriptional regulatory element can be used for either type of retrovirus...infect humans, many mammals, and even the avian population. Some adenoviruses are known to cause respiratory...induce a lower inflammatory response within hosts. Relative to the other viruses discussed in this article...

...mouse lines, you can access and manipulate an even further defined population of neurons. For example, say... workhorse adeno associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes...expression, detecting neuronal signaling, or manipulating any other genetically controlled function. By...Fig 3B). These specific neurons could then be manipulated (e.g., activated or inhibited) by any genetic...

... on the properties found in the literature or tabulated data that summarizes properties of fluorescent...) respectively. The theoretical brightness is calculated by multiplying EC by QY (EC*QY, sometimes normalized...several important experimental conditions that are related to the imaging strategy and the sample. Practical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...the assay is depicted in figure 1. The strict correlation between the fluorescent protein of interest and...

...splitting up a sample into thousands of physically isolated partitions (such as droplets or wells on a chip...edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for “functional...wild type target in each sample can be quickly calculated (details in [2]) and can be used to quantify ...few thousand cells from single wells of a 96-well plate without prior quantification. Comparatively, mismatch...AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base genome-edited human iPS cells without...

...engineers regularly use the platform, why scientists are using the platform, and tips for getting the most...from Nature Jobs Blog While Twitter is a popular platform for scientists, it’s not the only tool in a scientist... Facebook. For each of the three social media platforms, there’s some statistics on what percent of scientists...given you tips for using various social media platforms, and 3) helped you fit social media into your ...

... promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself ... vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these ...deletions of other regions, such as noncoding RNAs or regulatory elements, to examine their function. Bacteria... of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for multiplexed...gRNA Multiplexing Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR...

...aren’t, so repeated cycles will favor accumulation of the latter products. As noted above, a single cycle...repair, as there is no requirement for a repair template (sister chromatid or homologue) or extensive DNA... repair should take less than an hour, thus a population of cells constitutively expressing a targeted...NHEJ-dependent repair (“pop-in” insertion) provided a template containing compatible overhangs is available. ...

...watch my webinar, “Not Networking 101-Building Relationships for Success”. Choose 1-2 tactics to try. One...be a bit random because you never know which relationships will take off, or who your new contact will ...current organization but also important to build relationships outside your organization. The mentor in your... doing it again next month?” Some mentoring relationships grow naturally out of friendships or work interactions...other ways they can help you grow. Mentoring relationships can last for an hour or for a year. Some click...

...low abundance targets. Check out our post on Biotinylation to learn more about biotin and its use in molecular...groups from cysteines, or carbohydrates from glycosylation (Dennler, et al., 2015). However, there are ...Endogenous carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such ...2015) Antibody Conjugates: From Heterogeneous Populations to Defined Reagents. Antibodies 4:197–224. https...The Basics of Western BlottingPlasmids 101: Biotinylation Resources on Addgene.org Unconjugated antibodies...

...non-selective macroautophagy of the cytosol leads to accumulation of Pho8Δ60 in the vacuole, where it is cleaved...structures based on morphology. Autophagosomes are relatively easy to identify: double-membraned structures... GFP allows for tracking the localization or accumulation of pre-autophagosomal structures, autophagosomes...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive...induce autophagy, red but not green fluorescence accumulates in the vacuole. See Figure 2 for an example of...

...better salary increases that those in less densely populated areas. This assumption does not appear to hold...not getting a raise. Meanwhile, in less densely populated places like the University of Kansas and the University...say, salaries are showing surprisingly little correlation with cost of living at the location of the institution...important step forward in helping the postdoctoral population continue to pursue academic work, and to enhance...scientific enterprise by reducing the postdoctoral population which keeps growing despite a lack of available...

...assembly By Eric Perkins Though it’s easy enough to contemplate DNA in terms of its sequencing, in vivo studies...targeting a specific chromatin feature (e.g. a methylated histone such as H3K27me3), a linker between these... are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes. The speed and...used to bring cargo, such as protein or DNA, to manipulate function in receiver cells. There are over 40... G-baToN provides versibility in tracking and modulating the interactions between cells.      Tang et...

...crosslinking agent, a signaling ion, and an allosteric modulator. Because of its importance, scientists have developed...understand how this metal ion is trafficked and regulated. In this blog we will explore some of the most...and organs noninvasively and target a defined population of cells (e.g., a neuron subtype) and specific... calcium. It has been applied to mark active populations of neurons in vivo to study circuit activity.... allows scientists to focus on specific cell populations of interest. Many of the calcium sensors described...

... to specific sequences, causing targeted gene regulation. This can be used to activate transcription during...generation” CRISPR activator. While it requires a relatively simple construct, it exhibits modest levels of...activation experiments. In cases of multiplex gene regulation (activating multiple genes at once), however,...construction: it relies on antibody chains, which are relatively large and are not expressed consistently throughout... WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression...

...can spread between hosts through horizontal (or “lateral”) gene transfer. Horizontal gene transfer can occur...specialized degradative capabilities are often tightly regulated to be active only in the presence of the target...rapid spreading of genetic material through a population of cells. Cryptic plasmids: microbial mysteries...organization doesn’t necessarily reflect genetic relationships or plasmid incompatibility. Plasmids may also...2004). Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics...