...amino acid) AviTag sequence in a protein and adds biotin to a lysine residue within the AviTag. However,...modification of the purified target protein with the vitamin biotin. The interaction between biotin and the .... Avi-Tag plasmid for improved biotinylation selectivity A common strategy to biotinylate proteins involves... cannot be well-controlled. The lack of site selectivity typically diminishes the apparent affinity for...relatively low efficiency of biotinylation, at least in vitro. Our solution was to exploit the best of both worlds...carboxyl-terminus of the fusion protein includes an AviTag and a 6X HisTag for affinity purification (see ...ligase will biotinylate the lysine residue in the AviTag. As a side note, we found that adding a TEV linker...

...alpha2-AR. Selectivity Norepinephrine and dopamine are similar molecules, and cross-reactivity is a very...without biosensors to directly visualize their activity. In 2019, the first generation of norepinephrine...But this first generation of tools had limited sensitivity and speed and were only available as green probes...Kagiampaki et al. found nLight is suitable for in vitro, ex vivo, and in vivo experiments, and nLightG was... norepinephrine uptake throughout the brain. Sensitivity In ex vivo studies on mouse brain slices, nLightG... and nLightR showing significantly increased selectivity for norepinephrine over dopamine. All three sensors...to high concentrations of dopamine. This cross reactivity will need to be accounted for in your experimental...

...are critical: (1) connectivity to other data sources used in research; (2) connectivity to other tools used... each of these kinds of connectivity facilitates reproducibility. Connectivity to other data sources The...ecosystems and act as data silos because they limit connectivity with other tools and resources. Thus, it’s difficult... used in research; and (3) connectivity to open source data repositories. To illustrate this, let’s take...principles and has emerged as a byproduct of connectivity. Between 2012 and 2017 RSpace was developed ...have been implemented by providing support for connectivity to external data sources, tools used in research...data stored on institutional and lab servers. Connectivity to other tools used in research Just as researchers...

... the video below for an example of two-photon intravital microscopy from PLoS Pathogens - Kamenyeva et... technique and the in vivo live-cell imaging (intravital imaging) studies it’s enabled are revealing the...rapidly than any other cell type in the body! Intravital imaging powered by two-photon microscopy has ...of pathogen-immune cell interaction relied on in vitro studies where it was difficult to apprehend the ...nodes, brain, liver, gut and skin. Two-photon intravital microscopy. The video above depicts the infiltration...Technical challenges of two photon microscopy Intravital imaging is an easy way to understand what is ...fluorescence) are also useful tools to use with intravital imaging. These tools allow one to study the kinetics...

...its activity to produce light, often with a luminometer or scintillation counter, and the activity measurement...high-throughput screen using luciferase activity as a measure of signaling activity. Multiple response elements are...detection of the reporter protein or its enzymatic activity is measured. The effect of promoters or enhancer...ideally would have low background signal, high sensitivity and of course be quick, accurate and safe. In... lower background levels and improved signal sensitivity since photons are simply measured – they are ...Not Required Signal Strength Lower Higher Sensitivity Higher Lower Background Lower Higher Post-translational...sequence, resulting in a decrease in luciferase activity. This experimental protocol can readily screen...

...intensive and quite time consuming.   As Cas9/gRNA activity is crucial for successful CRISPR editing experiments...scientific community for efficient real-time Cas9 activity assays. Recently scientists have created CRISPR...reporter for assaying CRISPR/Cas-stimulated HDR activity) The Madison lab created SRIRACCHA in 2017 to ...expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio, edited cells can...SRIRACCHA enables scientists to both quantify Cas9 activity and sgRNA efficiency as well as enrich for mutants... fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome...are trying to target play a large role in Cas9 activity and efficiency (Sander and Joung, 2014). Thus ...

...Markus W. Covert. "High-Sensitivity Measurements of Multiple Kinase Activities in Live Single Cells." ...intermediate messengers. Optical control of kinase activity can provide greater spatiotemporal resolution ...system, light is used to turn “on” and “off” MEK1 activity by attaching two dimerizing photoswitchable Dronpa...its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK...you can precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling...it’s difficult to create short pulses of kinase activity. Zhou et al studied the negative feedback on endogenous...phosphorylation by creating a short pulse of psRaf1 activity. They did this by exposing psRaf1 to a short 2...

...dip in plasmid request activity around March. A rough estimate shows that activity level in March was around...the effect of COVID-19 on worldwide scientific activity. However, this type of open-ended question is ...plasmid requests from Addgene, as a proxy for global activity in our particular scientific subfields.  Plasmid...of the parameters we infer, since scientific productivity is better interpreted as the rate of scientists.... Values are the inferred percentage of normal activity (the average rate of unique PIs/labs placing orders.../15/20 (the supposed end of lockdown), request activity appears to return to normal. The model fit these... compatible with an infinity-fold reduction in activity due to COVID-19 to zero. Thus, depending on when...

...polymerase. SP6 polymerase has a high processivity. When used for in vitro transcription, the promoter drives...Typically contains a minimal promoter with low basal activity and several tetracycline operators. Transcription... in dicots, less active in monocots, with some activity in animal cells. Ubi General expression mRNA...Description Expression Additional considerations T7 in vitro transcription/ general expression Promoter from ...but requires T7 RNA polymerase. When used for in vitro transcription, the promoter drives either the sense...through varied inducer concentrations.  Sp6 in vitro transcription/ general expression Promoter from ... temperature sensitive cI857 repressor. T3 in vitro transcription/general expression Promoter from T3...

...Transdifferentiation in vivo or in vitro in vitro Both in vitro and in vivo Epigenetic marks Complete...redifferentiation into the cell type of choice in vitro is known as reprogramming [1]. The process can be...the earliest examples of transdifferentiation in vitro where treatment of mouse fibroblasts with 5-azacytidine...original somatic cells [41]. The in vivo and in vitro methods are similar except that the vectors carrying...reprogramming process as they are propagated in vitro as part of the process. This opens up a wide range...be to dedifferentiate cells from the patient in vitro then correct the damaged gene in the resulting iPSCs...PMID: 22632979. 14. Sasaki, K., et al., Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent...

...controlling CRISPR. To bring greater control to Acr activity as a biological tool, Niopek and colleagues turned...have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018). They accomplished... AcrIIA4. In the dark, AcrIIA4 inhibits CRISPR activity.   With the addition of blue light, the terminal...fusion protein inhibitor CASANOVA for “CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA4...2018. PubMed PMID: 30377362. Studying neuronal activity using the fluorescent voltage indicator Archon... Historically, scientists study the electrical activity in the brain by inserting electrodes into the ...of proteins for one that can report electrical activity via imaging. They took the light-sensitive protein...

...2020). It has increased CBE activity and reduced RNA off-targeting activity compared to ABEmax. In an alternative...to off-target editing based on Cas9 off-target activity.  Find CRISPR base editing plasmids at Addgene...). For each base editor, they observed editing activity with a minimum efficiency of ~50% and confirmed...TadA from ABE7.10 without increasing off-target activity (Richter et al., 2020). This is important because...ribonucleoprotein delivery decrease BE3 off-target activity Komor et al., 2017 BE4 and BE4-Gam; AID, CDA1...evolution of adenine base editors with increased activity and therapeutic application. Nature Biotechnology...efficient cytosine base editor with low off-target activity. Nature Biotechnology, 41(5), 673–685. https:/...

...detect PKA activity can be generalized to measure phosphatase activity, second messenger activity, and protein-protein...faster assembly and disassembly dynamics, higher sensitivity to light, and better distribution of clusters...biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes Jin Zhang... imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence fLuctuation...Dronpa. The authors tracked protein kinase A (PKA) activity at the plasma membrane in living cells using a...different time points, the authors tracked protein activity while also capturing information about spatial...

... make this STEM activity fun,” but rather “let’s see how we can make this fun activity relate to STEM....their expertise with the kids through some fun activities. If your school doesn’t have a Math & Science...parents from any background, it really helps if activities are run by parents who can answer some of the... a lot of kids, you’re going to want a lot of activities (try for a dozen!). The best approach may not...STEM.” Figure 1 shows the activities we had for my school’s Math & Science Night. There are plenty of places... places to get ideas for science activities. I did some crowdsourcing on Twitter and got lots of suggestions...Steam Powered Family  Biology and Chemistry activities from Quatr.us Foldscopes Steve Spangler Science...

...your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method for...selecting the guide RNA, validating its efficacy in vitro, and using it in mouse embryos to generate gene ...supplied by the Zhang lab. This simple website invites you to input the target region (approximately 500bp... many previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation...Validated gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers...in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene editing in mice Now ...  in vitro screening...

...the approaches that can be used to determine selectivity of an antibody for flow cytometry experiments...well as a pragmatic approach to determining the selectivity of antibodies for other sample types and for ...For these reasons, it is essential to test the selectivity of your antibody. But testing is not one size...your experiment. An antibody that demonstrates selectivity for cell surface labeling may become less selective...knockdown to enable robust assessment of antibody selectivity. Additionally, it can be a real challenge to ...protein level at the timepoint studied Off-target activities of the RNAi Correctly interpreting your data...protein expression makes confirmation of antibody selectivity even more challenging. Correlation with RNA or...

...balance maximizing on-target activity while minimizing off-target activity, which sounds obvious but can...100nt window downstream of the TSS gives the most activity. Thus, a given gene will only have a dozen or ...the method of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect...unwise to make conclusions on the basis of the activity of a single gRNA, and thus diversity of gRNAs ...approaches. Avoiding off-target effects The off-target activity of gRNAs is important to consider. While the basic...mismatches that can nevertheless still lead to activity has been established, and can be used to identify... indicates that the consequences of off-target activity, at least for the experimental conditions used...

...CasPEDIA feature. Activity features: CasID CasPEDIA will display both the cis and trans activity of each entry...enzyme: Nuclease properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability ...strands of its dsDNA target in cis and has no trans activity. AsCas12a, on the other hand, generates staggered...manually enter details to refine your search (trans activity, multiplex ability, gRNA design, etc.). With this... an evolving database, and all researchers are invited to contribute to it! The CasPEDIA Consortium will...update entries as research progresses, but you are invited to reach out to suggest new entries or even new...

... protein activity and have important roles during oxidative stress. To monitor this activity, Pedre Pérez...biosensors that detect new redox metabolites are vital for researchers to better understand the processes...assessed cFos expression. She will explore the connectivity patterns of these cells and the functional contribution... will allow us to better understand how neuron activity changes following opioid withdrawal, providing...excitatory and inhibitory neurons whose function and connectivity are poorly understood,” says Qiu.   Qiu developed...both optogenetics and chemogenetics to study the activity of parvalbumin (PV)-expressing neurons, neurons...

...retained the on-target activity of wt Cas9 while decreasing off-target activity at known off-target loci...displays comparable on-target activity and similar or decreased off-target activity across the sites tested ...effects of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and testing...Created with BioRender.com. To decrease the electropositivity of the HNH/RuvC groove, Slaymaker et al. made...combination mutants for their on-target and off-target activity at multiple genomic locations in HEK cells. Further...