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We narrowed to 581 results for: Vit

Showing: 61 - 80 of 581 results

  1. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

    Type
    Blog Post
    ...switch and turn off CRISPR activity. The ability to turn off CRISPR activity is an important step towards...thing that was missing: a way to shut off Cas’s activity. The concern is that the longer Cas remains active...edits to occur. Although methods to switch on Cas activity using light or drugs have been developed, the ... the labs found that anti-CRISPR prevents Cas9 activity in HEK293 by reducing editing from ~30% in control...proteins are highly diverse but most block CRISPR activity one of three ways: Inhibit DNA binding Example... Anti-CRISPR proteins (Acr's) can block CRISPR activity in many ways such as inhibiting crRNA loading,...experiments 1. Reduce off-target effects Prolonged Cas activity can increase the chance of off-target editing ...

  2. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...rapidly detect nucleic acids with high selectivity and sensitivity from patient samples. Find the DETECTR...nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at...level. The Zhang lab has adapted natural RNase activity of the Cas13 protein to develop and optimize the... the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2) (Gootenberg...CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (C2c2) was... your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or RNA from a sample... SHERLOCK to detect DNA or RNA with atomolar sensitivity and single base pair mismatch specificity.  SHERLOCKv2...

  3. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...for its role in molecular biology, biotin (Vitamin H or Vitamin B7) is first an essential coenzyme that ...determined to be due to a deficiency in Vitamin H later renamed Vitamin B7/biotin. Egg white injury would plague...biotinylation. This 13 amino acid sequence is termed AviTag. Using AviTag, one can label a protein of interest with...bacterial fusion proteins Biotin is an essential vitamin for life Although biotin is most commonly known...bacteria and some fungi, thus mammals must obtain the vitamin through external sources. Humans absorb most of... biotinylate macromolecules of interest both in vitro and in vivo. Biotinylation reagents Biotin’s exposed...Scientific's Biotinylation Reagent Selection Tool. AviTag and BirA Proteins can also be biotinylated by exploiting...

  4. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ...to control Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity. B) OptoTIAM (magenta)... light-sensitive ion channels used to control activities like neuronal signaling — think of a mouse with...there’s a lot more to optogenetics than neuronal activity. Researchers have designed a variety of photoswitchable...severing. A) Schematic of Opto-katanin constructs and activity. EB3N-VVDfast-iLID (anchor) and SspB-mCherry-p60...microtubule severing and has very low dark-state activity and toxicity. All of that means this light-powered...called SuperPLDs with 10, 30, 50, and 100 times the activity of the original, generated using directed evolution... with high efficiency in mammalian cells and in vitro, opening up a wide realm of possibilities. The CRY2...

  5. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...can interfere with activity. The resulting system was coined as CRISPR–Cas9 activity switching via a novel...stemmed from the goal of reducing off-target Cas9 activity. The use of nickases, prime editing, anti-CRISPR...targeting specificity or reduce the duration of Cas9 activity.  The field of optogenetics is renowned for enabling...such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR...NLSx3-CRYPHR-p65 - it had the lowest background activity in the dark state and highest fold induction at...where light is required to reconstitute enzymatic activity. Photoactivatable genome modifications by NHEJ...low background and high fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system...

  6. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ... chance of cross-reactivity Higher level of background stainingLess sensitivityLess specificityLimited...specificityIncreased chance of cross-reactivity Sandwich High specificityHigh sensitivity Longer protocolIncreased...moleculesReduced chance of cross-reactivity Less sensitivityLess specificityLimited availability of conjugated... ELISAs for their accuracy, specificity, and sensitivity (a good ELISA can detect picogram quantities ...procedure removes one potential source of cross-reactivity.   While the method is simple and saves time,...Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary antibodies...secondary antibodies can also be a source of cross-reactivity.  Sandwich ELISAs In the most common type of...

  7. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...and predicted on-target activity was balanced with predicted off-target activity when selecting guides....lower off-target activity, but it could also be caused by a reduction of on-target activity. However, there...-target activity, Morgens et al drew some generalizable conclusions about off-target activity, demonstrate...off-target cutting without a reduction of on-target activity, but it’s unclear if this holds true in genome-wide...sites that lack open chromatin marks, DNase hypersensitivity, or are in an enhancer, transcription factor...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated...guides with high GC content have greater off-target activity. Short gRNAs have lower off-target cutting and...

  8. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ...between recipients.   In vitro prediction of drug immunogenicity  The goal of in vitro prediction of immunogenicity..., 2020).  Evaluating a drug’s immunogenicity in-vitro helps researchers understand a drug’s safety and...depending on the person. However, in silico and in vitro assessment of immunogenicity can help evaluate risk...silico prediction algorithms fail to capture. In vitro evaluation of ‌therapeutic immunogenicity is typically...induction (Groell et.al, 2018). Thus, microscopic in-vitro models can be built that contain various components...components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of therapeutic...efficacy.  Better together Combining in silico and in vitro approaches enables researchers to develop safer ...

  9. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ... light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated...to blue light, this channel activates neuronal activity by opening and allowing positively charged ions...developed many optogenetic tools to control cellular activity with exquisite precision using only light. Optogenetics... tools, can control everything from electrical activity to gene expression, and even CRISPR based gene... optogenetic tool causing a change in cellular activity or gene expression. Experimental output is measured...(rhodopsins) either excite or inhibit neuronal activity upon exposure to light within a specific range...downstream signaling cascades (reviewed in Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS...

  10. In Vivo Biotinylation of Bacterial Fusion Proteins

    Type
    Blog Post
    ...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...

  11. New Norepinephrine Indicators: nLightG and nLightR

    Type
    Blog Post
    ...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...

  12. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ... 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...

  13. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ...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...

  14. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    ...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 ...

  15. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    ...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...

  16. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...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...

  17. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    ...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...

  18. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

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    Blog Post
    ...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...

  19. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    ...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...

  20. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...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:/...
Showing: 61 - 80 of 581 results