... and image analysis software are highly sensitive to detecting light signals not visible to the human ... that the resolution of light microscopy is limited by the wavelength of light and, practically speaking...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...well-characterized marker proteins tagged with FPs to highlight various subcellular structures. Please see Figure...visualize subcellular structures. For example, you might use your fluoresent protein fused to a signal sequence...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...PMC3074624. 5. Bindels, Daphne S., et al. "mScarlet: a bright monomeric red fluorescent protein for cellular ...

...antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context. Sci...However, it was (and still is) infeasible to generate highly specific antibodies for every potential new target...interest with unrelated peptides that already had high quality antibodies available - aka peptide tagging...tagging.  Key to this strategy was the existence of high quality antibodies for well defined peptides. Munro...the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino ...sounds less than ideal.  Unexpected connections Right around the same time that Munro and Pelham were ... (1) Locate a promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope...

... an MRI, you know that magnetic fields can pass right through our tissues unimpeded. Andrew and Maria ...for a magnetic response among other proteins that might serve as a starting point for developing useful ...Andrew and Maria explained, “to our surprise and delight,” the very first protein they tested — E. coli ...coli expressing good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet...team was careful to test the possibility that this might just be an artifact and eliminate sources of interference...expressed and purified. Although a ~1% change in signal might not make a very powerful tool, it was promising ...enhanced for future MagLOV-based tools, just as light-induced conformational changes are the basis of ...

...Every student about to graduate shares the same nightmare: the question “So, what comes next?” For those...education or ending a Master’s program, the answer might be to continue in academia and pursue a PhD. However... such as Zoom, to determine if a program is the right fit for you without the financial and logistical...to around twelve professors and heard back from eight. These emails don’t have to be long; the idea is...you a chance to get to know the faculty that you might be working with for the duration of your program...that will help you determine if the program is the right fit. Reaching out to PIs also means doing plenty...close to your computer with things you want to highlight in the interview is another way to make the virtual...

...keep in mind that UV light only kills microorganisms that it strikes directly. UV light exposure is also ...ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help...also harmful for eyes and skin so keep the light off when the hood is in use. Wipe the hood with a disinfectant...source of contamination for your experiments, the bright side is that you also have the power to take the...morphology: Shape and size matter You should have a light microscope close to the hood so you can view your... morphologies and doing a quick check under the light microscope will help you identify bacterial and ...are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...

...verify loss of minicircle RNA Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections... Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome integration Insertional...reactivation, residual expression Lentivirus Integrating High Low Efficient and Stable Genome integration Insertional...transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free. Slow and inefficient...reprogramming efficiencies ranking as low, medium or high is based on the reprogramming efficiencies reported...pMXs or pMSCV, infect dividing cells, and have a higher efficiency of generating iPSCs compared to other...integrate or replicate. Their small size leads to higher transfection efficiencies and they tend to express...

... cell or neuron type may not work well for a neighboring cell type, even within the same brain region ...Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained against dopamine...frustrating and often months-long quests to find the right AAV for your system or to successfully replicate...sharing of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve ...and that it often provides incredibly valuable insight into one’s work. As a community, we must therefore... descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating vector... do you have sitting in computers and notebooks right now?, I’m willing to bet the answer would be along...

...from Addgene right on our plasmid pages and provide a citation that you can put straight into your reference...our website using the search bar found at the top right of every page. You can search for plasmids by name... number, or keyword. Although an initial search might give you many results, you can narrow your search...search results using the filters found on the right side of the search page. A great breakdown of our search...special collections pages. These pages serve to highlight plasmids that can be used for specific purposes...Every quarter we release a Newsletter where we spotlight some of the plasmids that have come through our...page - each day we showcase a new plasmid that you might otherwise miss. We hope this feature will draw attention...

...serum. Harrington et al suspected GeoCas9 might have higher stability since prior research had shown that...zebrafish, frogs, some bacteria; but not thermophilic (high heat loving) bacteria. Until recently the only available...s SpCas9. These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria...approach only works for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles... RNP activity or longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of...tested the ability of GeoCas9 RNPs to be incubated at high temps but still maintain activity following heat...temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the ...

...Every few months we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...Improved voltage indicator: JEDI-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody...indicator (GEVI) called JEDI-2P. Using a custom, high-throughput screening platform to apply saturating...10.1016/j.cell.2022.07.013.   Novel class of light-gated potassium channelrhodopsins by: Mike Lacy ...Xue and John Spudich identifies a novel class of light-gated potassium channelrhodopsins, named Kalium ...2022). The authors show that HcKCR1 and HcKCR2 have high selectivity for K+ over Na+ and rapid photocurrent... optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na+ and other cations...

...neuron-optimized viral vectors, and high-throughput screening approaches. Here, we’ll highlight these recent advancements...least, more straightforward than generating cDNA for each gene isoform individually. To highlight this advantage... The Day Lab used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA...targeted anywhere in the genome, it is relatively straightforward to use CRISPRa to selectively target these ...-optimized lentiviral vectors can be found here. High-throughput screening strategies Although the tools...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in our repository...different genomic loci — in some cases, over 20-fold higher editing than PEmax. The authors hypothesized that...immunoblotted against vinculin as a loading control (right panel). Image from Addgene. These antibodies... subthreshold voltage fluctuations, while having high fidelity during action potentials of fast-spiking...j.neuron.2023.03.009. PMID: 37015225.  Simple and highly specific targeting of microglia with AAV By Alyssa...human IBA1 promoter (hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated...hIBA1a promoters in mice. B) Quantifications showing high microglia specificity of GFP expression for the ...

...upper right) and a mixed population of B cells and dendritic cells (DCs) in the lower middle right. DCs...generated often looks like the graph on the left, it might make more sense once you realize events are plotted...plotted on an invisible Cartesian plane (right.) Each event is plotted by the amount of signal read from...cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive for...another two are B+. Our A-B+ cells are in the lower right corner.      Figure 2: This flow plot has... (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell, instead of fluorescent...DCs), mDCs and pDCs. Let's follow it from left to right.  First, SSC and FSC are measured to differentiate...

...FRET signal you should choose the highest quantum yield donor, the highest absorbing acceptor and fluorophores... similar brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP...that case the cross-talk is high and your background signal may be higher than the signal generated by... the acceptor (linking the quantity of absorbed light, at a given wavelength, to the concentration of ...that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry...the donor is excited by a specific wavelength of light and the signal is collected by using emission filters... of the donor is increased. This method is straightforward and quantitative, but it is destructive and...

...insertional effects in the genome. (Bottom panel, right) A highly expressing RUBY plant is a vibrant red/magenta...transparent tissues, or in green tissues is slightly darker or slightly brown. Photos by Andrew Willoughby ...something new has been added to the palette. RUBY is a bright red pigment reporter whose expresion is visible...RUBY as a selection marker The creators of RUBY highlight its use in crop plants under field conditions ... further experiments.     Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following floral...from the same plant beginning to germinate. (Top right, second panel) Transformed seedlings can be selected...

...listen, and an ability to remember the right stories at the right time. Like many of the posts in our ...be considered in the right, and one in the wrong. If you arbitrate, one party might get exactly what they...I first realized I might have some minor skills in conflict resolution after college, when I discovered...inclination for this skill that you could strengthen. The right set of transferable skills, coupled with your technical...primary role is customer support, but there are eight other teams who still retain a customer support ...be wary of anyone who is actually looking for a fight. But you also can’t be the type of person who avoids...or invalidated. Both compromise and arbitration might remedy short-term substantive conflicts, but they...

...and it emits light that peaks at 508 nm. The protonated state, however, does not absorb light at 488 nm.... efforts have increased the brightness of these genetically encoded light emitters (i.e. Takai, 2015 and... enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another mutation (A206K) is necessary...of the available green fluorescent proteins emit light. The pKa of fluorescent proteins can be accurately...that are engineered to bind specifically and with high affinity to fluorophores. Examples are the FlAsH...fluorescence overlaps with autofluorescence The cyan light that is used to excite GFP, may also excite several...shifted to longer wavelengths. Therefore, the use of bright red fluorescent proteins (e.g. mScarlet) or infrared...

...total protein are compared. While a higher slope indicates higher sensitivity, linearity is the most important... quantification of a western blot — and why you might want to be careful relying on so called “housekeeping... simply reports which POI band is stronger. The right gel has exactly the same POI bands as the left gel...plot of protein abundance versus band intensity (right). Created with BioRender.com. On this gel, the... the protein of interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the...under your experimental conditions. Otherwise, you might want to consider a different loading control. Assumption...accurately quantified Most housekeeping proteins are highly expressed in cells. This means that at sample concentrations...

...illuminates the passing cell with a laser and detects the light scattered by that cell at a low angle (FSC) or large...voltages to observe your cells on a flow plot, you might have to do some testing or ask experienced colleagues..., morphology, and structural complexity.   Height, Width, and Area Within FSC and SSC, there are ...are three parameters that are being measured: height (H), area (A), and width (W). The different measures...few events at the end of your gating pipeline, it might be worth going back and loosening the FSC/SSC gate...Figure 2), single cell events will show up in a straight line one the plot, while duplicates deviate from..., it can depend on the type of stain whether a tighter or looser gate makes sense. Here, luckily, the ...

...the PCR process, you need a high-fidelity polymerase. There are many high fidelty polymerases on the market...Cornell University. Site directed mutagenesis is a highly versatile technique that can be used to introduce...SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis...site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate...Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction site “A*” results ...rather than a linear product (see figure). Template A high purity plasmid prep significantly increases the ... target site, can identify these duplications (slightly larger bands sizes relative to the original template...