We narrowed to 740 results for: NSI

...fluorescence intensity remains. The first issue is that, in several cases, the fluorescence intensity decay ..., fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017...experiment is performed that measures fluorescence intensity over time. In order to predict how fluorophores...proteins and by comparing how the fluorescence intensity changes over time, the photostability of the fluorescent...photobleaching rate does not depend on fluorescence intensity or protein distribution. Therefore, photostability...analogous to radioactive decay). Consequently, the intensity decay is often described by the t½, which is the... it is necessary to know how the fluorescence intensity evolves over time. The second issue is that, in...

...help with transitioning out of academia (because so many scientists are making this transition). Another...have experience and knowledge to help scientists transition out of the academic sphere into a different meaningful...not always as effective in mentoring for this transition. Second, a coach will help you set goals and ...successful outcome and the good ones take this responsibility seriously. As I make my rounds speaking at ...However most coaches who focus on scientists are sensitive to this and have coaching plans that are financially...here are a number I have come across for you to consider. Feel free to add other suggestions in the comments...scientific backgrounds. For a stated purpose of transitioning a scientist from academia to industry, you might...

...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events.... Z. (2024). A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events... tags for single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies...collaborators recently created ASAP5, a faster and more responsive genetically-encoded voltage indicator (GEVI) ...ASAP5 selected for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and...regimes with fast activation kinetics and high responsivity near the resting membrane potential, ASAP5 is...the formation of Gα-GTP by BRET. The biosensor consists of a YFP-tagged G protein and a NanoLuc® (Nluc...

... problem. It is estimated that mycoplasma is responsible for up to 60% of the cell culture contamination...contamination (Uphoff, 2002). Mycoplasmas are considered to be one of the simplest and smallest bacteria. The absence... source of culture is irreplaceable or highly expensive). Detecting mycoplasma contamination Detection...Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting mycoplasmas...noticeable color change. This method has high sensitivity but low specificity (Degeling, 2012). Bioluminescence...result interpretation might be difficult. The low density of mycoplasmas, contamination by other bacteria...you keep all the required equipment and reagents inside the laminar airflow before and while you are working...

...Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells. Methods Mol...based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021 Sep;118(9):3499-...At its core, harvesting recombinant antibodies consists of separating antibodies from media, cells, and...this method, beads are incubated with the cell suspension. Antibodies bind ligands on the beads and a magnet...alongside your ladder and a protein standard. The density of the antibody bands can be measured against the...multiple contaminating bands or a few strong ones, consider increasing the number of washes performed during...conformational changes in the antigen or lower detection sensitivity of the assay. Where possible, it can be useful...

...system both favors antibiotic sensitive bacteria and prevents sensitive bacteria from acquiring resistance...system. These CRISPR antimicrobials successfully resensitized a beta-lactam resistant population to the antibiotic... may result in plasmid loss and antibiotic resensitization, or may also cause cell death. Image from Beisel...et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant bacteria (Figure 2). The...Bacteria that receive this phage are now antibiotic sensitive, but lytic phage-resistant. When the lytic phage...killed, antibiotics can be used to target the sensitive population. Yosef et al.’s proof-of-concept work....   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics. A lysogenic phage infects...

...in yeast with Rosella, a pH-sensitive fluorescent biosensor A pH-sensitive fluorescent biosensor-based...-emission biosensor Rosella consists of DsRed connected to a pH-sensitive variant of GFP (SEP) by a 9 ...Virus (WNV) induced cell death. The Wu lab library consists of 77,406 individual gRNAs targeting a total of...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...plasmids to the scientific community. This collection consists of plasmids containing genes from a crowd-sourced...scientists use GMAP, there will be a continuous expansion of compatible backbones, promoters, and genes ...all under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression...

...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID enzyme, Lee and Cheah...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID and APEX2 ...trafficking within and between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j....

...Cell lines should be routinely subcultured at densities that are low enough to prevent confluency but ... in lower lentiviral titers. Another factor to consider is the age of your culture post thaw. In general...can be subtle and may include reduced saturation density, decreased rates of growth, or clumping. To ensure... and resource consumption due to the high cell density of the culture. In addition to benefitting your...lower titer virus. In such cases you may want to consider concentrating your viral prep. Storage can also... start to a successful infection, one point to consider is how the virus is being titered. Most titering... that routinely produce lentivirus may want to consider testing a variety of cation and serum sources ...

...experiments a bit more challenging. With these considerations in mind, when planning an experiment users ...transgene, and downstream applications should all be considered when choosing the correct system. Recently, ...volume. Consequently, you will end up with a considerably lower volume than with the unconcentrated prep...experiment requires a certain volume of virus, consider scaling up by either increasing the number of ...while the PEG precipitation method is relatively inexpensive and does not require an ultracentrifuge, PEG ...lentivirus, it is time to titer. One important consideration when titering a lentivirus is the cell line ...transducing a target cell line. One should also consider the specifics of the titering transduction experiment...

... example, while gene knockouts have long been considered the “gold-standard” for LOF studies, the high...complementary to the dsRNA (Wilson and Doudna, 2013). RNAi consists of two non-exclusive gene silencing pathways: ...data generated from both methods did a more comprehensive gene list emerge that accurately reflected the...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based... of the RNAi trigger requires a few initial considerations, such as the starting material (DNA-based vector...target sequences are concatenated. hpRNA has been extensively used in plant research and many different constructs...further rounds of RNAi. This process, known as “transitivity,” calls into question any claims about specificity...

...the tool, and develop new applications for magnetoresponsive proteins. Motivated by curiosity Although...Maria dreamed about the possibilities of a magnetoresponsive fluorescent protein. Controlling an optogenetic...Frank Hayward et al., 2024). The fluorescence intensity decreased whenever the magnet approached the sample... in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response...self-labeling FlavinTag made a reliable one-component magnetoresponsive system that could be expressed and purified...cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations...with other engineering approaches and that magnetosensitive tools can be developed from other fluorescent...

...specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the ...thus tracking the localization of that biomolecule inside living cells. FPs classified by the emission color...range, there are other traits that need to be considered when choosing an FP: Unique categories of fluorescent...distance between the two FPs is <10nm, and should be considered when labeling proteins that interact. Indeed,...Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For instance...experimental parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent...experimental parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter...

...biologists were no longer beholden to chemists and (expensive) synthetic fluorophores. Add a dash of DNA with...to small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances...requires more photons. For example, moving from transient overexpression of protein fusions to gene-edited... HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that...fluorogens (Figure 1e). Stains – These labels consist of a fluorophores conjugated to molecular species...can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling tag systems...groups (Figure 2b), which can preclude their use inside living cells. We recently discovered that incorporation...

...tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2...dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids created for ...plasmid backbone that was designed specifically for transient expression (find some useful backbones here) or... or from viral transduction. While not ideal, transient expression of viral constructs is a useful tool...cell line. When expressing a viral construct transiently care needs to be taken to ensure that the proper...differ in two ways. Firstly, 2nd generation systems consist of an envelope plasmid and a packaging plasmid ... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...

...come up with a list of things to consider as you make this transition. Of course, a “desk job” could entail...employee who's coming directly from grad school. The transition has been a bit rough for her (3 weeks in and ...there are any resources that can help with the transition.” The lab-to-desk-job culture shock is a real...scientist colleagues have as well. Making the transition from the bench can be jarring for the unprepared...resources out there for scientists making this transition. In this installment of the Addgene career guide...What should you expect during the lab → office transition? I’ve already provided a few counterpoints to...likely happen eventually. If you approach that transition dreading the change, the adjustment period will...

...develops an enhanced propensity for movement and activity called locomotor sensitization. This enhanced movement... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...ve previously discussed some of the general considerations for performing behavioral tests, but now we...seeking drugs’).  What’s involved in locomotor sensitization? Previous research has shown that regions such... glutamate transmission leads to locomotion sensitization. (for review see van Huijstee & Mansvelder  ...suggested inhibitory mechanism of locomotor sensitization and might open new ways for a novel therapeutic...subset of excitatory neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically...

...training schedule that helped me transition easily. Training and responsibilities Through this training, I was...The application process When I first saw the comprehensive job description for this co-op, I was immediately...me a full description of the tasks I would be responsible for, in comparison to the other positions listed...for the independent work on the QC team. I was responsible for helping other scientists on the team ensure...sequencing QC work    In addition to my responsibilities on the QC team, I was able to explore other...uses something called “core hours”. Core hours consist of the time between 10 AM and 3 PM, when most meetings... the “9-5” was not as scary as it seemed. The transition into a traditional work environment was aided...

...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers... with fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks...Optogenetics functions through opsins which are light sensitive proteins. Opsins used in optogenetic studies are...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...inhibition: Hyperpolarization Conversely, when the inside of the neuron becomes more negative it becomes ...differing peak absorption wavelengths and light intensity requirements of different opsins, studies have...

...A major limitation of APEX was its low sensitivity. Sensitivity could be increased with higher expression... avidin, proximity labelling can capture weak/transient protein interactions as well as be used for both...promiscuous biotin ligase (Kim et al 2016). BioID2 consists of a humanized biotin ligase from A. aeolicus,...cell.   Find BioID2 plasmids here APEX2: A more sensitive ascorbate peroxidase for proximity labeling A ...aggregation. The Ting lab hypothesized that the low sensitivity was due to suboptimal folding or stability. Thus...identified a mutant with significantly increased sensitivity that did not affect the function or efficiency... assemble when in close proximity. Split-BioID consists of a split BirA* molecule that can reassemble ...