... This is an important downside of the technology and should always be considered when interpreting results...oligomerization of fluorescent proteins is a concern. The propensity of fluorescent proteins to oligomerize is known...brightness, photostability, maturation time, and acid sensitivity of various fluorescent proteins (Botman et al...coded by the multiple cloning site and therefore consist of random amino acid sequences or (ii) designed... an inert, unstructured peptide and therefore consisted of glycines, serines and other small, non-aliphatic...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...fluorescent protein fusions There are several choices to consider before generating a fusion protein, e.g. linker...

... Morgridge Institute for Research in Madison, Wisconsin. It is commonly cited that approximately one-third...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...The development of phosphoserine incorporation Extensive genomic sequencing of prokaryotic lineages revealed...specialized elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide...available through Addgene, but we have worked most extensively worked with and optimized Sep-OTSλ and can recommend...the deletion of release factor 1, the protein responsible for terminating translation in response to the...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...

... other ligands - almost A to Z! - from ATP to urotensin. The MTRIA sensors will be useful for investigating...displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR. Image from... response kinetics and increased brightness, sensitivity, and photostability under two-photon illumination...proteins (miRFP670nano and miRFP720) and the Ca2+-sensing domain from Twitch-2B (Matlashov et al. 2022). ...plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium ...LiLac has improved response size and reduced sensitivity to pH and calcium compared to previous lactate...470-495 nm) and is capable of local temperature sensing with sub-millisecond resolution and sub-degree ...

...integrating a SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore...organoids By Ashley Waldron Organoids are three-dimensional cultures that mimic aspects of organ morphology...enzyme biosensors provide direct, specific and sensitive detection of the plant hormones strigolactones...the receptor which modulates the fluorescence intensity of the cpGFP without altering that of the internal...fluorophores, rather than on the fluorescence intensity, it provides a direct and quantitative detection...receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for...

...temperature drops, single intensity should smoothly increase as the DNA amplicon transitions from single-stranded...detect a single copy of a transcript. But this sensitivity can sometimes cause as many issues as it solves...separate thermocycle allows you to measure the intensity of signal over an entire cycle. Since signals ...double-stranded DNA, when temperatures are high, signal intensity should be low, reflecting the presence of primarily...assumption that there is a smooth, single-step transition between ssDNA and dsDNA. This is not true for...one (Biassoni, 2014). At the bench As a highly sensitive technique, qPCR needs a steady pair of hands to...important as precision for relative quantification. A consistent 5% positive error across the plate is fine; a...

...should be consistent across all samples on the gel and should be observed as a band of consistent size and...These systems, however, require you to purchase expensive system-specific sandwiches.   Figure 2:...vendors will show that the antibody binds to transiently overexpressed protein, however, they should also...includes publications that cited the antibody, consider reviewing the materials and methods sections of...and density present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent...you require precise quantification of a protein, consider using an alternative method such as an enzyme-...

...ultimately responsible for outcomes. Be ready to support your team and take responsibility if things go...series on Management for scientists. Once you are responsible for managing others you will only be successful...you become a master at delegating tasks and responsibilities. The manager is not expected to DO all the...experience? Do you think you are giving this responsibility to someone who can do the job. It is good to...sure it goes well? As with most managerial responsibilities, good communication is at the heart of delegating...boundaries. Where are the lines of authority, responsibility and accountability? What types of issues do...

...resultant cells can either express the plasmid DNA transiently (similar to bacteria) or incorporate the genetic...ever-versatile fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For... for the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection...want the plasmid depleted, but still want to use transient transfection methods. Since there are no "natural... generations. Stably transfected cells may be considered an entirely new cell line from that of the original...plasmid contains a cassette that will make cells sensitive to a toxin). In the table below we focus on positive...causes strand scissions 100-400 ug/mL *Not comprehensive.   ** In eukaryotes.   ***The concentration ...

...or qPCR-based methods to greatly increase the sensitivity of detection (14-20). LDR has also been extended... do not read specific base sequences, but are sensitive to distortions in helix shape (22). Large purine... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful... that extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology...In this method, substrate pools were prepared consisting of one target (template) strand and four upstream...Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem. 77 (2008): 313-338. PubMed...JS, et al. "A high-throughput assay for the comprehensive profiling of DNA ligase fidelity." Nucleic acids...

... cells are sensitive, it might be more difficult to get them to grow out. The only downside is that it.... In theory you could use densitometric analysis to quantitate the intensity of the bands on your gel ...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...followed. If you have never used this assay before, consider following their tips, including: Before digesting...

...GCE4All: Making Genetic Code Expansion Accessible Addgene's Genetic Code Expansion Collection...protein tags. The basics These tags most often consist of an enzyme that has been mutated to block its...CP appearance at the filament tip from (a). c) Intensity traces at the tip of the filament from (a-b), ... effects on actin filament length (lower). d) Intensity and filament length traces for a different event...of organic dyes or specialized ligands is more expensive than letting the cells make a fluorescent protein...Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science, 281(5374), 269–272. https:... W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research, 44...

...turned to optogenetics. Through the use of light-sensitive protein domains, they have developed a system .... They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop region ...brain. But this process is time consuming and expensive. Thus scientists have turned to genetically encoded...electrical activity via imaging. They took the light-sensitive protein QuasAr2 and systematically mutated it....localization, has high signal:noise ratio, high sensitivity, responds quickly, is photostable, and is compatible...interaction (PPI) studies in plants that rely on transient transfection systems using Agrobacterium (such...Grefen and Blatt, 2012). The 2in1 Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different...

...recently developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to...and their research.   Abhi Aggarwal: Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate ...new version of the biosensor is faster and more sensitive than its predecessors. Currently, Aggarwal, and...Research Center (DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based biosensors for live cells.... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...

...within 10 nanometers of each other, or measure the tension in the helical structure of spider silk, or the...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...Förster resonance energy transfer by monitoring sensitized emission in living plant cells." (2013). PubMed...linker sequence between the donor and acceptor is sensitive to a change in the environment which alters the...processes and signaling cascades, quantify mechanical tension (a molecular “spring”) (Meng et al., 2008), and...animals (Hirata and Kiyokawa 2016), and molecular tension microscopy (MTM), which could monitor physical ...Charlène, and Borghi, Nicolas. "FRET-based molecular tension microscopy." Methods 94 (2016): 33-42. PubMed PMID...

...for optogenetics. There are several important considerations with fabrication of the fiber optics needed...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...will be erratic, opsin activation/inactivation inconsistent, and behavioral results impossible to interpret...sequentially increasing fineness helps to maintain a consistent stream of light. Test your implant by connecting...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the...: closed/light receptive, open, closed/light insensitive. If you deliver a continuous stream of photons...optogenetics we’ll delve into some of the important considerations when performing optogenetics behavioral experiments...

...methodological and experimental limitations and considerations? I recently spent two months in the lab of ... at NIDA and some of the practicalities and considerations you may not find in the materials and methods...use optogenetics The most important question to consider is what exactly do you want to use optogenetics...are also available. Picking your opsin  You must consider whether you wish to stimulate or inhibit neuronal...include Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which...potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride ion pumps, activation of which results...breeding, and maintenance of a mouse line is also an expensive and time-consuming endeavor. Finally, expression...

...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...cameras and image analysis software are highly sensitive to detecting light signals not visible to the ...human eye and are indifferent to subjective color intensity perception. Open source image analysis software... and well-executed experiments require some consideration of FP properties. It is important to look out...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

...such as mice, which are less prolific and more expensive per individual to maintain. Also in contrast to...heart defects and cancer. These mutants are used extensively to study development and disease. Additional ...implemented in zebrafish. Developmental biology insights using lineage tracing in zebrafish To highlight...lineage tracing approaches that led to important insights in developmental biology. Microscopy-based lineage...embryo, where similar processes occur over days inside the mother, the zebrafish is an ideal system to...their long three-month generation time and labor-intensive maintenance compared to invertebrate model systems...highlight how these advantages can translate into insights in developmental biology, and advances in this...

...library/produced your lentivirus. Since viruses are sensitive to freeze/thaw cycles, we include a 1.3 mL aliquot... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...transducing cells at a concentrated seeding density while centrifuging. Spinfection transduction efficiency...optimization of many variables, such as seeding density and reagent concentration.  Screening time Phew...place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...Libraries- A nice overview of what to consider when using pooled lentiviral CRISPR libraries. Protocols from... Waxmonsky NC, Swanson LE, Fan M. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...can be a time-intensive endeavor. That is why we also included an analogous set of transient constitutively-expressed...on the other hand, is a widely available and inexpensive imaging modality with good tissue penetration... be easily expressed heterologously by bacteria inside the body. So we screened gas vesicles gene clusters...bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors was overlaid with the anatomical B-mode...Emerald GFP. These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293... using the most reliable method, which is the transient cotransfection of the mARG plamsids into HEK293T...