...Renilla luciferase variant fused to an FP; when supplied with a luciferase substrate, the luciferase transfers...bioluminescence resonance energy transfer (BRET). Photons emitted by a Renilla luciferase variant are used to excite...problems of low luminescence By itself, Renilla luciferase has a low quantum yield, and when transiently...Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent technology...need for an excitation light source. Instead, a luciferase substrate like coelenterazine is supplied in ...technique’s versatility and adaptability to many different contexts. Improving Nano-lanterns In 2015, Takai...eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate furimazine. In addition...

...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which... acceptor fluorophore. The difference for BRET is that the energy transfer is between a luminescent donor...number of different experimental approaches. They first assessed the activation of different members of...response to different agonists. These studies showed the promiscuity of GPCRs for different G protein transducers...neurotransmission. But with approximately 950 different human genes encoding GPCRs (Takeda S et al., 2002...a vast number of signaling cascades, inducing different cellular responses. One of the biggest advantages...measure the physical association of combinations of different Gα, Gβ, and Gγ subunits. When inactive, these ...

...control many cellular processes such as differentiation, proliferation, and apoptosis. In humans, mutations...Protein A-Renilla luciferase and the “prey” proteins are tagged with Firefly luciferase. The bait and prey...variable availability of enhancer sequences in different chromatin states. Alexander Stark and colleagues... were deep sequenced to identify the gRNAs that confer protection to WNV. From this screen the authors...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...consists of a variety of plasmids containing many different promoters, RBS’, coding sequences (CDS), and terminators...To facilitate innovative cancer research, the Reference Reagents Group of the NCI RAS Initiative, led ...

... variables.    Serotypes Serotype refers to the different protein compositions of AAV capsids, which... a virus enters the cell. Different serotypes utilize a number of different receptors, co-receptors, and...burdensome in the brain where many different cell types, or even different phenotypes of the same cell type...working in several different systems or want to transduce a large number of different cells. Promoters ...doi.org/10.3389/fnmol.2014.00076 References and Resources References Aschauer, D. F., Kreuz, S., & Rumpel...validation and optimization. Although there are different ways to ultimately achieve success, we find it...validation first.  AAV-mediated gene expression can differ extensively depending on the experimental system...

... However, the two different approaches allow for optimized strategies for differing amounts of target ...urea-containing lysis buffer. Alternatively, the protein can also be eluted using a low pH buffer such as 0.1M ...for other antibody topics.     References and resources References:  Björck L, Kronvall G (1984) Purification... appropriate for most situations.  A slightly different method is desirable if the antibody is expected...Immobilization is the key to immunoprecipitation and simply refers to the process of anchoring an antibody, often ...eliminating the risk of the primary antibody interfering with downstream assays. This is particularly ...than those of agarose beads, despite the size difference. Magnetic beads are particularly advantageous...

...target primaries produced in different species and to label targets with different colors. Multiplex immunofluorescence...Speaking of different colors, having a palette of secondary antibodies in a few different species and ...dimensional epitopes. Different sample processing steps can alter epitopes in different ways, so antibodies... a successful IF experiment.    References and resources References Im K, Mareninov S, Diaz MFP, Yong...immunohistochemistry (IHC). All three of these terms refer to the use of antibodies to visualize a target within...essentially just more general terms that can also refer to assays using non-fluorescent visualization methods...indirect immunofluorescence There are a couple different approaches to be familiar with when talking about...

... IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation and had ...number, generally very small, and can contain different neurotransmitters. Using genetically encoded fluorescent... brain.  Figure 1:Schematic diagram of different FingR constructs and experimental results. Image... of the cell. By combining vectors expressing different fluorescent proteins targeting inhibitory or excitatory... proteins system for Caenorhabditis elegans By Jennifer Tsang Fluorescent proteins (FPs) traditionally...The team validated this tool by targeting six different genes whose protein products have distinct localizations...Figure 2: Split-wrmScarlet labeling proteins with different subcellular locations. Image from Goudeau et al...

...blot Run your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize...before rinsing with wash buffer, as dry PVDF won’t evenly re-absorb aqueous buffer. Be sure to check the ...from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the best...abundant. If the abundance of your targets isn’t too different, consider antibody affinity: stronger binding ...should not compare the strength of bands from different rounds of detection. Finally, stripping and reprobing.... Unlike PVDF, they can be returned directly to buffer without a re-wetting step. Important: do not dry...so you’ve dried your membrane and returned it to buffer, you’ve run and imaged your western blot with your...

...worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities depending... Resources References Shaw, G., Morse, S., Ararat, M., & Graham, F. L. (2002). Preferential transformation...make the virus. Viruses can be produced in many different cell types, spanning both prokaryotic and eukaryotic...However, some viruses can be produced in multiple different cell types, including plants, mammals, and insects...improve your current methods, understanding the differences can help you make an informed decision. Mammalian...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...standard to do only one passage (transfection). This differs from typical insect cell preparations, which we...

...expression. Gene isoforms refer to mRNAs generated from the same gene but with different untranslated or coding...activated cell sorting (FACS) to isolate differentiated from undifferentiated cells at the end of the screening... to study neuronal differentiation, it could also be applied to the differentiation of other CNS cell ..., we’ll highlight these recent advancements and offer commentary on their application to neuroscience ...straightforward to use CRISPRa to selectively target these different transcriptional start sites in order to better... expression of Bdnf resulted in hundreds of differentially expressed genes between Bdnf I-overexpressing...which transcription factors promote neuronal differentiation (Liu et al., 2018). The pooled nature of this...

... immune system make different isotypes of antibodies that recognize many different epitopes of the target... and protein G each have different affinities for IgG subtypes from different animals so it’s important...successful next experiment!     References and Resources References Ascoli CA, Aggeler B (2018) Overlooked... cover polyclonal antibodies, one of the many different types of antibodies available (others you’ll encounter...polyclonal antibodies from the serum There are a few different ways the antibodies can be isolated from the serum...by an animal are specific for the immunogen. A different type of purification can be performed to isolate...experiments where antibodies raised in several different species are present. One common example is using...

...Castle et al., 2014). Differences between AAV serotypes may be also linked to differences in viral uptake by...these different AAV vectors into a brain region, the AAV particles will infect neurons at different ratios...architecture, in particular the connections between different brain regions. For example, communication between...2015). Directional flow of information between different parts of the brain is mediated via individual ... for degradation. These two pathways also use different cytoskeletal machineries to facilitate transport...while anterograde transport relies on kinesin. Different rates of speed for various forms of retrograde... or have been genetically engineered to have differences in their capsid proteins. For a brief overview...

...Your AAVancement could be next!   References and Resources References Braun, C. J., Adames, A. C., Saur...allows for a wide variety of screens, including interference, activation, and knockout. Most CRISPR screens...CRISPR screens can be carried out using a few different methods — in vivo, in vitro, and ex vivo. The .... The go-to in vivo option AAV has become the preferred delivery method for in vivo CRISPR screens. AAV...titers equal to those achieved with lentivirus but offer an improved safety profile, customization, and enhanced...mouse tissues. Safety AAV and lentivirus have different safety profiles that often affect which is chosen... cause bad reactions in mice. Customization AAV offers a level of customization that is unmatched by other...

...antibodies are all of different species/isotypes and that each secondary has a different color fluorophore...a comment below. Happy imaging! References and Resources References Andrews, N. P., Boeckman, J. X., ...be familiar with immunofluorescence (IF, often referred to as immunocytochemistry (ICC) when the sample...mixture of secondary antibodies, and image all the different colors in one session – fast and easy! Just make...Trimmer Lab, using recombinant antibodies with different isotypes. (A) Neocortex labeled with anti-pan-...make sure you are using primary antibodies from different species or isotypes, so that the secondary antibodies...to align the images of each labeled target as different color channels and analyze the results. Depending...

...looking at a bunch of different strategies with CRISPR-Cas9. There are so many different variants and strategies...will help them to understand how different mutations lead to differences in the symptoms experienced by ...competition for researchers working on rare diseases, offering them the chance to win some of the latest tools...with the condition to reveal some “outstanding differences.” They now have some theories on the underlying...had the opportunity to meet a few patients that suffered this disease,” Aguilar said. “Once you have that...Institute and Addgene will make a substantial difference in pushing the work forward. Xeroderma Pigmentosum...lot of those plasmids so that we can try many different things in parallel.” Alternating Hemiplegia of...

...demonstrated that different deaminase and UNG enzymes had different sequence preferences, which they defined...outcomes of their various CGBEs at different target sequences and in different cell types, available at www....non-target strand.” To avoid confusion, we usually use different terminology when discussing base editors, like...up to 68% at the most favorable target sites. Differential cytosine editing between bacteria and mammalian...sites. But the same strategy in E. coli produced different results. GBEs seemed to follow the same initial...editors Now that several labs had shown that preferential C → G editing was possible, the race was on ...creating BE variants with other Cas9s that recognize different PAMs. Their computational tool for predicting ...

... with a T-DNA (transfer-DNA) region. During infection this T-DNA region is transferred into the plant....track, we switched to luciferases to more easily quantify the effect of the different treatments on transformation...method. Transfer your plant into the dark 16 hours before the transformation. We routinely transfer our plants...the T-DNA region with the gene of interest is transferred into the cells. But for C. roseus, Agrobacterium... lot of time. Don't re-invent the wheel. Test different leaf ages Usually younger leaves work better. ...GUS::tMAS to confirm transformation success. Luciferases are great quantitative reporters and use a substrate...substrate to produce bioluminescence. Luciferases usually have shorter mRNA and protein half-lives then...

...applicable to the rat [6]. Technical differences include the ferrule implants and patch cables. One huge...intracranial self-stimulation (ICSS) and place preference. The lab I work in (the Kreek lab) focuses on...behaviors to test reinforcement are ICSS and place preference.   Historically, ICSS was done by implanting ...nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement but ...halves that are distinguished visually through different patterns and the animal only receives stimulation...). If a circuit is reinforcing, the animal will prefer to spend most of its time in the chamber in which...Comparison of c-fos amongst animals that receive different types of stimulation may help to refine and optimize...

...find lots of different plasmids containing the same protein of interest tagged with different fusion proteins...you the flexibility to choose different colors or ligands for different experiments without having to ...intracellular recordings, switching between different colored ligands at different time points.   Figure 3:...or HaloTag have been shown to perform differently for different ligands (Wilhelm et al., 2021). While ...things up for your next experiments! References and resources References Bottanelli, F., Kromann, E. B., Allgeyer...proteins for different experiments over the years — a green fluorescent protein, a red fluorescent protein... Combining orthogonal self-labeling tags on different proteins of interest (or one self-labeling tag...

... transposition.  Target site: Different transposons insert at different DNA sequences or target sites....has several different applications (mutagenesis screens, transgenic animals, gene transfer). The discovery...specific locations in the genome.   References and Resources References Feschotte C, Pritham EJ (2007) DNA...commonly used in the lab While there are many different types of transposons, DNA transposons are most... vectors.   Let’s take a closer look at three different transposon systems adapted for use as research...Sandoval-Villegas et al., 2021). Sleeping Beauty’s preferred target site for integration is TA dinucleotides...piggyBac, Tol2 has a weak consensus sequence for its preferred target integration site: TNA(C/G)TTATAA(G/C)TNA...