...well since the N- and C-terminal residues of the fluorescent protein are relatively close together (figure...fluorescent protein is at the C-terminus of the POI or that the POI is at the C-terminus of the fluorescent... design and engineering of fusion proteins is relatively straightforward. However, generating a fusion...fluorescent protein. Thus, scientist began using relatively long linkers between the protein of interest ...dimerization (Shaner et al., 2008 - supplementary figure C). However, these assays are qualitative. The Organized...structural information available, it’s clear that the C-terminus of fluorescent proteins derived from Aequorea...biosensors (yellow cameleon 3.60, EPAC, and Galphai), the C-terminus and N-terminus of the donor fluorescent protein...

...machinery making an original C-G pair converted to T-A or reverted to the original C-G by base excision repair.... Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation by base editors. Pathways...suitable distance from the base (C/A) to be edited. But does this mean a target C or A much further from the...editing efficiency based on the location of the ‘A’ relative to the protospacer adjacent motif (PAM). ABE7.10...edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018) (see CBE...binding with DNA, the CBE deaminates a target cytosine (C) into a uracil (U) base. Later the resultant U-G pair...original genomic A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision ...

....38853 Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of Interactions among bZIP and Rel Family Proteins...of interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would...necessary because split FPs can associate, albeit at relatively low frequencies, without being brought into close...visualized makes it amenable to studying PPIs under relatively physiological conditions when compared to FRET...information, you can fuse each FP fragment to the N- and C-terminal end of each PIP and empirically test which...R, Björklund M, Ahr D, Ahmed AW, Spinelli L, Brun C, Basler K, Merabet S (2018) Generation of a versatile...doi.org/10.1101/pdb.prot076497 Kim Y, Jung JP, Pack C-G, Huh W-K (2018) Global analysis of protein homomerization...

...Y (T or C), X (any base), or C, respectively. gGBE, gTBE, gCBE Glycosylase-based G, T, or C base editors...transversion editors: CGBEs, or C → G Base Editors. CGBEs first convert a cytosine (C) to uracil (U), just like... a more specific A → C base editor (ACBE), they inserted the deaminase into the C-terminal domain of Cas9...pyrimidines (C ↔ T).   Base transversion Base change from purine (A or G) to pyrimidine (C or T) or vice-versa...type of base editor invented. CGBE C → G Base Editor Converts C → G by fusing a uracil DNA glycosylase..., or C, respectively, to be replaced with another base. References Chen, L., Hong, M., Luan, C., Gao... Zhang, D., Zhang, S., Li, C., Wu, J., Randolph, P. B., Sousa, A. A., Qu, C., Zhu, Y., Guan, Y., Wang,...

...Tobey, I. G., Moncada-Reid, C., Eid, F.-E., Walkey, C. J., Ljungberg, M. C., Lagor, W. R., Heaney, J. ...Ravindra Kumar, S., Adams, C. D., Yang, D., Wang, T., Wolfe, D. A., Arokiaraj, C. M., Ngo, V., Campos, L....Gong, Y., Ng, C., Nammour, J., Maiz, D., Dujardin, S., Ramirez, S. H., Hudry, E., & Maguire, C. A. (2019)... Poterba, T., Boutros, C. L., Balazs, A. B., Daneman, R., Bloom, J. M., Seed, C., & Deverman, B. E. (2019...AAV-PHP-B, it can be produced with high viral yields relative to AAV9. At lower viral titers than AAV-PHP.B ...when paired with the ubiquitous CAG promoter, is relatively biased toward L5 and inhibitory neurons and astrocytes...C57BL/6J and BALB/cJ mice through LY6C1, with relatively even distribution of target gene expression (...

... works well is that the N- and C-terminus of GFP itself are relatively close, thereby minimizing the disruption...Also, in cases where the protein of interest is relatively small, adding GFP may alter diffusion rates. ...Although bioluminescent probes are usually dim relative to fluorescent probes, recent engineering efforts...Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus, but this... standard practice to attach the GFP to the N- or C-terminus of another protein. This may, however, interfere...consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence (-CaaX). Both modifications...when GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the alternative...

...an ortholog in C. elegans making it ideal to study basic molecular biology processes. C. elegans are also...PMCID: PMC5525645. Mello, Craig C., et al. "Efficient gene transfer in C. elegans: extrachromosomal maintenance... model organism for scientists as they have a relatively short generation time for mammals - the time ...that are laid externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists...lab to study neuronal development (Brenner, 1973). C. elegans are small, transparent worms that have a ...life cycle and large brood sizes. The majority of C. elegans are also self-fertile hermaphrodites making... Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout their...

...80 °C. Titer All titering is performed on lentiviral preparations that have been stored at -80 °C and ...of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins...SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed...Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. ...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...Preparations are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or ...preparations are then aliquoted, frozen, and stored at -80 °C. Concentrated lentiviral preparations (“-LVC” catalog...

...the order they appear in the plasmid and their relative position to the insert. Example, if you have a... immediately followed by its position (number) relative to the start Methionine (Met) followed by the ...resistance, b) the promoter that drives the insert, and c) any other selection markers (for use in other cell...pBACKBONE-Flag-hGene If there was also an EGFP fused to the C-terminal of your insert you would list it after the...

...j.cell.2021.09.018 Choi, J., Chen, W., Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza,...efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning that most..., J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A.,...Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations...pegRNAs (Yan et al., 2024). The team fused La to the C-terminal end of PEmax to produce PE7. PE7 significantly...expression ~ ~ ~ ~ ✓   ✓ SV40 and c-Myc nuclear localization sequences Improve translocation...References Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy...

...mesothelioma that affects the lining of the lungs is relatively well known, there are a variety of lesser known...disease are unclear. Dr. Di Schiavi plans on using C. elegans as a model to identify SMN1 interacting partners...effects of smn1 disruption. As part of his work in C. elegans, Dr. Di Schiavi will use Addgene plasmids...these genes, CRISPR to disrupt their function, and C. elegans overexpression plasmids to upregulate their...research funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...heart health. These are amazing advances that will surely save thousands if not millions of lives, but many...therapy for peritoneal mesothelioma and thereby relieve suffering from this little-known disease. You can...

...-018-0219-4  Yu C-C (Jay), Barry NC, Wassie AT, et al (2020) Expansion microscopy of C. elegans. Elife...physically separates molecules while maintaining their relative positions, allowing you to resolve individual ...accessible imaging equipment, ExM protocols utilize relatively inexpensive reagents and are readily adoptable...brain slices, which are easier to expand than, say, a C. elegans (Damstra, et al. 2022; Yu, et al. 2020). .../doi.org/10.1038/nbt.3625  Truckenbrodt S, Sommer C, Rizzoli SO, Danzl JG (2019) A practical guide to ...they want to understand the ultrastructural relationships that influence everything from cell division...optimization and careful execution, but they cannot be entirely eliminated. It is recommended that you image samples...

...independent blots. B–C) Graphs showing the relative intensity of the β-actin (B) or total protein (C) on the membrane...needed for relative quantification of a western blot — and why you might want to be careful relying on so ...can you accurately compare different lanes? In relative quantification, the POI bands are normalized to...quantification in panel B is not especially linear. In panel C, two different methods of quantifying total protein...linear. Note the different y-axes between panels B and C, indicating that β-actin is less sensitive as well...your protein of interest (POI). This approach is reliable, but it’s overkill in many cases. Often, you may...protein concentration in your sample would directly correlate to protein abundance on your membrane, which would...

...Zhou C, Gao N, Rao Z, Li H, Hu X, Li C, Yao X, Shen X, Sun Y, Wei Y, Liu F, Ying W, Zhang J, Tang C, Zhang...method is its construction: it relies on antibody chains, which are relatively large and are not expressed...generation” CRISPR activator. While it requires a relatively simple construct, it exhibits modest levels of...Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki...To target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of ...in their transcriptional activators: some methods rely on fusion proteins while others re-engineer components...that it requires a fusion protein, rather than relying on a two-component system dependent on gRNA design...

... 79628 (C) Cv (capsid) N and C 79633 (N) and 79641 (C) NS1 (var: W98G) N and C 79635 NS3 C 79637...79637 NS2B C 79636 NS4A N 79640 NS4B C 79639 NS5 C 79632 (N) and 79631 (C) PrM N and C   Detecting...environment and relies on the fact that enhancers function independently of their position relative to a transcription...parts involved in altering gene expression are relatively well known, particularly in bacteria, the process...strains (5). MutL is highly conserved in distant relatives of E. coli so it can be used in a broad range ...The key to the biosensor lies in pH: DsRed is relatively pH-insensitive, while SEP fluoresces at cellular... and normalized to assess interactions. Higher relative firefly luciferase activity in the Co-IP compared...

...variation. Confirmation of sequence can be done from relatively cheap plasmid sequencing. While rAbs are monoclonal...to run a western blot. It allows you to detect relatively low levels of antigen, while providing flexibility...assay design. Conjugated secondary antibodies are relatively cheap and easy to source, making it easy to switch... not available in a pure form, you can perform relative quantification, which measures protein amounts...particular antibody, this truly does provide a relative, not absolute, readout. Normalization Overall ...purified protein not always available Calculate relative quantification Normalizing to ubiquitously expressed...length of your assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most...

...Biddle, M. S., Alende, C., Fotouhi, M., Jones, C., Ayoubi, R., Southern, K., Laflamme, C., Virk, H., NeuroSGC...T. M., Brown, C., Bandrowski, A., Virk, H., Edwards, A. M., McPherson, P., & Laflamme, C. (2023). Scaling....154034.1 Laflamme, C., McKeever, P. M., Kumar, R., Schwartz, J., Kolahdouzan, M., Chen, C. X., You, Z., Benaliouad...populations and to study relative protein expression. Its reliability will be entirely dependent on the selectivity...and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is, an antibody..., H. S., Biddle, M. S., Smallwood, D. T., Weston, C. A., Castells, E., Bowman, V. W., McCarthy, J., Amrani...Biddle, M. S., Wright, A. K. A., Sousa, J., Weston, C. A., Chachi, L., Roach, K. M., & Bradding, P. (2019...

...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s. These...Compared to BE3, BE4 offers a 2.3 fold decrease in C→G and C→A products as well as a 2.3 fold decrease in indel... Hong, M., Zhang, D., Luan, C., Zhang, S., Wu, H., Gao, H., Bai, S., Li, C., Ding, R., Xue, N., Lei, Z... W., Levy, J. M., Yeh, W.-H., Zheng, C., Newby, G. A., Wilson, C., Bhaumik, M., Shubina-Oleinik, O., Holt...non-homologous end joining (NHEJ), HDR occurs at a relatively low frequency, especially in nondividing cells...) and adenine base editors (ABEs). CBEs mediate a C to T change (or a G to A change on the opposite strand...strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are base transition...

...You know qPCR is most commonly used to measure relative quantification of mRNA, which can be used as a...reported to work well in the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table... Accuracy is not as important as precision for relative quantification. A consistent 5% positive error...Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time...150 bp long Annealing temperature should be 60-65°C For qPCR, the important primer variable is the annealing... the primers.  Melting temperature should be ~60°C (between 50 and 65, with no more than 3 degrees between...curve will show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak...

..., Banh, L. M., Williams, C., Toh, M. S., Irby, M. J., Edwards, L. S., Lin, C., Owen, A. L. G., Künne, ... other Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved...contains all five of these Cas proteins, while type I-C Cascades do not contain Cas6. Regardless of the subtype...Resources References Loeff, L., Brouns, S. J., & Joo, C. (2018). Repetitive DNA reeling by the Cascade-Cas3...Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5....Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, P. D., Rotstein, T., Kohrs,...inversions and large deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding...