...substantial size of GFP may interfere with the activity or the localization of the fusion protein. Also... visualization of heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32....al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3 (2018): e0193705... proteins are also a better choice for thicker samples. Another solution to avoid autofluorescence is ...reaction and hence, there is no need to excite the sample. Although bioluminescent probes are usually dim...

...Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (...bio-His 51749 EFNB1-bio-His 51750 EPHB1-bio-His 51751 ESAM-bio-His 51754 FCGR2a-bio-His 51755 FURIN-bio-His...

...of the optogenetic regulator leads to spurious activation of the FRET sensor. Researchers have sought to...Here the bioluminescent donor is unaffected by activation of other fluorescent probes but can still transfer...as the excitation wavelengths needed would also activate the optogenetic accuators. The researchers took...NLuc requires 100-fold less protein to produce the same amount of light from an Fluc or RLuc reaction. In...protein that could be co-imaged with GFP using the same wavelength for excitation. The result was the cyan-excitable... K., et al. (5) imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs...and colleagues (8) were interested in using photoactivatable, optogenetic accuators to depolarize or hyperpolarize...

... This repeat region associates with the trans-activating crRNA (tracrRNA) which interacts with the Cas9...Modifications of CRISPR RNAs to improve Gene-Editing Activity and Specificity. JACS 144, 12584-12594 (2022).... when scientists talk about their precious RNA samples. RNA is inherently less stable than most macromolecules...CRISPR-Cas9 experiments – the “gRNA” – suffers from the same issues as your experimental RNA. To solve this problem...Another stabilizing sugar modification, found at the same site, is 2’-fluoro (2’-F), which substitutes a fluorine...protein and can increase on-target efficiency. In the same vein, locked nucleic acids or bridged nucleic acids...modification have overcome these woes with photoactivatable and photocleavable gRNAs. Photocleavable guides...

...variation in color depends on how transcriptionally active the genomic locus of integration is. The white ...TH17 cell differentiation by antagonizing RORγt activity. Nature. 472, 486–90 (2011). PubMed PMID: 21441909...recombination so that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct...variants of YFG because the transgenes are found in the same locus across fly lines (i.e. there won’t be positioning...

...use of high voltages, please use caution when activating pulse and follow all specifications described...gel extraction of either the original sample or the amplified sample, followed by reamplification of the...tens to millions of different plasmids in a single sample. They are often used for screening, barcoding, ...appear to contain sufficient DNA for use, pool samples, and continue with analysis. “Sufficient” DNA will...

...level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It is a Drosophila...turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences found in ...this mutant fly to one lacking the mutation in the same genetic background. In other cases, YFG or one of...

...structure can readily fold into transcriptionally active double-stranded form through intra-molecular annealing...expression patterns of AAV8 vectors with broadly active promoters from subretinal injections of neonatal...episomal and does not divide and replicate in the same way the host DNA does. Because of this, fast dividing...McCarty, D. M., Paul E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated...

...hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of interest ...heavy and light chain, each from a constitutively active CMV promoter. Constructs were then verified by ...detect multiple targets with different mAbs in the same experiment. The Trimmer Lab found that they could...

...fluorescent proteins (FP) require external light activation, you can’t use fluorescence to monitor processes...light used for excitation can cause unintended activation of the optogenetic system. Biosensors based on...external light source. Therefore, especially thick samples may not be appropriate for use with Nano-lantern...Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check Out Fluorescent...

...recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons.  PubMed...ability to conditionally turn off one gene and activate another. In the field of mouse genetics, FLEx ...between the sites. If a DNA sequence is floxed in the same orientation, the SSR will excise the DNA fragment...reporter with one loxP site and one lox511 site in the same orientation, and placing the additional loxP and...

...scientists and non-scientists alike, need to stay active and involved if we wish to make positive change...chair of the postdoctoral engagement committee and active member of the university's Postdoctoral Association...this year I had never taken part in any public activism. One of my favorite signs from the march read ...Deanna McLafferty is an editorial assistant and activist. Washington, DC, USA 38.9072°N 77.0369°W – Mark...campus speakers thoughtfully reminded us to use the same critical lens on our scientific community as we ...well-publicized, we might encounter hostile anti-science activists. However, I must say that I was supremely proud... nor would you mention my small error and at the same time pass over in silence his very large one” (http...

...subsequently destroyed by Argonaute-catalyzed nuclease activity or their translation disrupted through physical...the appropriate plasmid and dsRNA expression is activated through the  induction of T7 polymerase encoded...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for in...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...

...could in theory promote off-target binding. The activity of SpRY was validated in double stranded cleavage...assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic Acids Res. 2021 Sep...& Walton et al). Additionally, SpG Cas9 with the same PAM preference of NG was isolated by the Kleinstiver...several bps away from a desired site does not have the same devastating impact on efficiency as 10-15 bps away...

... derailing the show. The moderator must be very active at all times keep things moving and to prevent ...interesting to the audience. It is a bit of an art, but active moderation gets easier with practice. Up the audience...answers uninteresting, the panel members all say the same thing and one panel member dominates the discussion... for an intimate event, put the panelists on the same level as the audience. In my experience the best...

... the known pathway that were essential for the activity of different ligands. Such knowledge could be ... to other nucleases as well as inhibitory and activating approaches.  Many thanks to our guest blogger...G., et al. "Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9...validation screen using selected gRNAs extracted from the same or another library. These different approaches could...Listed you can also easily extract gRNAs for the same gene from several libraries, and identify those ...

...enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together very large toolboxes...the amount of transcriptional and translational activity a cell has and control how much it taxes the resources...problems. You couldn’t do it without repeating the same guide RNA twice. The second problem was the shape...

...Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be used...tube as shown below. For 8 samples prepare enough master mix for 9 samples. Component Volume 9X Volume...constant across samples. In the RRE example below, 2-fold serial dilutions of a sample were loaded in ...ddPCR Lentivirus sample data, RPP30 positive (green) and negative (black) droplets Sample ID Plasmid ID ... higher dilutions depending on their particular sample. This protocol was modified from the publication...Starting Thaw the master mix, primers/probe mixes and samples on ice before use. Wipe down all pipettes and surfaces...

...interest wanes off such that attendees are not actively participating, it may be time to consider ending...clubs where participants are passive rather than active and the meeting is often driven by one or two participants...itself A journal club should aim to have many of the same characteristics as a successful meeting: a clear...through the paper figure by figure. Some follow the same figure by figure organization with a different person...stay abreast of current trends in the literature, sampling a breadth of work from a field (perhaps even two...outside of one's own field in a structured and interactive way. Like any meeting, to be successful in serving...

...site of incorporation (Figure 1d). Fluorogen activating proteins (FAPs) – These modified antibody fragments...William Perkin. His discovery set off a flurry of activity and the majority of the classic small molecule...removing excess dye from the sample, which is particularly important for samples where washing is difficult...small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances lead to an ...reason is something called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore...of information one can extract from a biological sample is wholly dependent on the photon budget and pushing... desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...