...induced and quantified by measuring the amount of radioactivity in the culture supernatant (this is indicative...degraded) and it is slow. Alkaline phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline...to generate an active enzyme. Pho8 phosphatase activity or the molecular weight shift of Pho8 from its...regardless of its localization in the cell. SEP’s pH sensitivity means Rosella will fluoresce green unless it’...

...2020. PubMed PMID: 31937940. Detection of neural activity using a red fluorescent genetically encoded voltage...detect and output the voltage changes in neural activity when paired with appropriate fluorescent optics...combines the Cre/lox system with optogenetics. Cre activity is controlled by red light by splitting Cre and...bringing each terminus of Cre together to restore Cre activity. As soon as red light or PCB is no longer present...

... without causing high background due to cross-reactivity. When to use polyclonal antibodies One advantage...labs. Polyclonal antibodies also have a high sensitivity to the target protein due to their ability to...or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly useful... epitopes, increasing the likelihood of cross-reactivity with other proteins. As with any experiment, ...

...Why study neural connectivity? One of the early lessons many of us learned in biology is that the body...address this, molecular tools to map neuronal connectivity are widely used in neuroscience. In this post...emerged as powerful tools for visualizing neural connectivity (Nassi et al. 2015). The recombinant viruses...of a G-deficient rabies virus recombinant: an in vitro and in vivo study. J Gen Virol. 2000 Sep;81(Pt 9...

...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery ...how can a DNA-binding protein with no enzymatic activity cause demethylation?  Dividing cells depend on...Mattei AL et al. (2020) TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated...conserved C-terminal domain of TET1 to regulate TET1 activity and function in development. eLife 7:e34870. https...

...no-G PAM library to enrich for Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To...xCas9 in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells, finding xCas9...PAM flexibility might also increase off-target activity. Surprisingly, off-target effects across 5 gRNAs... as improvements at NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct NGG PAM NGA...

...majority have been shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both...alternative approach is to modulate Cas9’s nuclease activity by modifying the gRNA with which it is complexed...capability remains intact while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be...means of exerting tight control over Cas9 nuclease activity while eliminating the reliance on orthogonal Cas9...

...produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo/ssDNA requires incubation at 55...cells and one which diminishes the endonuclease activity of the enzyme. In vivo, only nascent NgAgo protein...can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA...does however rely on cloning into a vector or in vitro synthesis, which could be less desirable if one ...

...find people who are. I wanted to do fun science activities with the public, so I reached out to people who...what career suits you best I began viewing my activities as types of experiments. I searched for new opportunities...made me happy. Grad school can provide plenty of negativity, so I volunteered at a place that made me happy...recharged. The fun change of pace helped break up the negativity and allowed me to focus on the positive. I went...

...identified in Doench et al., 2014 to predict sgRNA activity. In this study scientists examined approximately...sequence features and adjustments that improved sgRNA activity and thus library performance. The Root and Doench...This library was designed to improve on-target activity while reducing off-target effects in human genomes...G., et al. "Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9...

...knockdown, it still displays substantial off-target activity. By mutating residues in ADAR2 that interact with...the RNA substrate resulted in 15% C-to-U editing activity. They then performed sixteen rounds of directed...ADAR2DD to further optimize cytidine deaminase activity. While RESCUE can perform C-to-U edits, the authors...East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and...

..., easily purified protein with easily measured activity, MDH is particularly amenable to in-class labs...   It is important to note that research activities in a CURE must be authentic – students should...give up the control embedded in “cookbook” lab activities (follow the recipe, and you get a predetermined...using a 6XHis tag, and compared their specific activities by measuring NADH production.   CUREs are...

...28b-Cas9-His from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by... advantage has allowed scientists to perform in vitro and in vivo CRISPR studies in experimental systems...approach using RNPs has recently been described in vitro, with the hope of eventually using this method for...enables efficient protein-based genome editing in vitro and in vivo." Nature biotechnology 33.1 (2015): ...

...been transformed. But caution: check for GUS-like activity of your tissue. Run a control with Agrobacterium...gene since plants can have endogenous GUS-like activity (Hu et al., 1990). We commonly use pSB161 - pL2...therefore good temporal reporters to measure promoter activity, for example. Useful positive controls could include...115. Hu, Ching-yeh, et al. "Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990...

...extracellular surface) which enables tracking of protease activity with a fluorescent protein--no affinity purification...tested different target sequences for improved selectivity and found that the best motif was from the Lck...commonly used for in vivo biotinylation via BirA and AviTAG. pMAK contains the signal peptide of MBP, followed...followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag. The MBP signal peptide allows the...

...Specific RNA cleavage (followed by non-specific RNAse activity in bacteria) Cas13a targeting with a single...the pool of nucleotides, the nonspecific RNAse activity of Cas13a becomes activated and the RNA reporter...LwaCas13a does not display nonspecific RNA cleavage activity in eukaryotic cells. They arrived at this finding...East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and...

...a little more complicated since basal neuronal activity in your brain region of interest could be low ...21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...PMC3150193. 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development and application...M., et al. "Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans." Nature...

...virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently...investigating HIV integrase mutants in the early 1990s (Leavitt et al., 1996), and since then, numerous variants...PMID: 18688286 PubMed Central PMCID: PMC2496964 Leavitt, A. D., Robles, G., Alesandro, N., & Varmus, H....

... of any of these phosphorylation events on the activity of a given protein? Classical methods for studying...non-phosphorylated recombinant protein and perform in vitro kinase reactions. Often, however, researchers lack...or have difficulties with kinase specificity in vitro. The establishment of a system in which purified...pSer/phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic mutation...

...infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived vectors are capable...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...have to be aware that there is often  background activity and it requires continuous administration of Dox...GFP encoded virus, and the reverse transcriptase activity assay uses qPCR to quantify viral RNA. All of ...