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...Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides to a substrate Flow...carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier...chemically. Enzymatic biotinylation occurs naturally in cells and is the process by which biotin is covalently...sequestering biotin from being absorbed. In 1940 Esmond E. Snell was able to purify the protein responsible for binding...the chicken, “Avian”, egg it was purified from. Snell later confirmed that avidin was indeed the culprit... for use in immunohistochemistry and antibody labelling. In these experiments, biotin is added to a primary... Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for biotinylating...

...Shcherbakova et al., 2016), as well as in four-color imaging (cyan, yellow, red, and near-infrared) (Qian...reporters and biosensors NIR reporters can tell us about cellular events via  fluorescence changes occurring...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...

...). The Heroes of CRISPR. Cell, 164(1), 18–28. https://doi.org/10.1016/j.cell.2015.12.041  Musunuru, K....birth. Within a seven-month whirlwind of developing cell and mouse model systems, testing base editing variants...reporting genome editing by CRISPR in mammalian cells appeared in 2013 (Cong et al., 2013; reviewed in...editor (ABE) followed based on similar principles (Gaudelli et al., 2017). These editors did not create double-stranded... The first ABE was about 50% efficient in human cells on average, but its effectiveness varied depending... the chromosomal location, sequence context, and cell type. Multiple labs worked to improve its effectiveness...and more flexible in their target preferences (Gaudelli et al., 2020; Richter et al., 2020).  Five years...

... Mixing the subclones within a single well reduced well-to-well variability, while the readout was able.... Nat Cell Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal...with one visual barcode per cell and up to 200-plex with two barcodes per cell. The researchers validated...long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for ... powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview...reduced risks of unwanted mutations in mammalian cells. Long sequences traditionally have higher risks ...multiplexing by: Rachel Leeson Multiplexed images in live cells remains a challenge due to multiple factors, including...

... N1 Yellow Mammalian Express a gene of interest fused to the N-terminus of Amber Amber C1 Yellow Mammalian... pCEP4YPet-MAMM Yellow Mammalian Expresses mammalian optimized YPet pYPet-His Yellow Bacterial Expresses...(FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. ... The student was of course talking about the now well-known FRET, aka Fluorescence (Förster) Resonance...measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions...suitable for studying interactions within living cells. By coupling fluorophores to proteins, pioneers ...directly detect protein/protein interactions in living cells. Since then FRET has also been used to measure conformational...

... For most kits, the steps for lysing cells and removing cellular debris look very similar. However, keep... Yes —beads, columns, plates, buffers 96-well plates, 8-well kit, automation-friendly Not mentioned Endotoxin-free...relative term — even armed with the highest-competency cells you will still struggle to get transformants if ...quantity) – Depending on the experiment, mammalian cell transfections can require quite large amounts of...your plasmid; separate plasmid DNA from unwanted cellular “debris”; and then clean-up the plasmid DNA (Figure...your bacteria to release the plasmid, separate the cell debris from the plasmid, and then clean-up the plasmid...and column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications...

...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...application of AAV is delivery of genetic cargo to a host cell. In order to make room for a gene of interest, AAVs...Serotype refers to the type of capsid (outer protein shell) used to package an rAAV vector; these capsids allow...allow the virus to infect (or not infect) different cell types with some specificity. There are a lot of ...

...be applicable to studies of cell cycle progression, epigenetics and cellular reactions to external stimuli...Weissman JS, Qi LS, Huang B. Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001. Pubmed. Find...LS, Weissman JS, Vale RD. Cell. 2014 Oct 23;159(3):635-46. doi: 10.1016/j.cell.2014.09.039. Epub 2014 Oct...labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, ...CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique...nuclear organization varies across the life of a cell, or how organization may be altered in disease states...dead Cas9 (dCas9) has many applications, the most well-known being transcriptional activation/repression...

...illumination of neighboring cells evokes action potentials (APs) in patched cells expressing wild type CoChR...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here...28646204. soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego ...wild type CoChR, soCoChR localizes primarily to the cell body with very little signal detected along the ...

...neuroscience where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...disease, neuroscientists need to be able to probe cells and circuits without disrupting normal brain function...it provides a noninvasive method for manipulating cell activity, especially neuronal firing. What is chemogenetics...drugs. Once activated, chemogenetic receptors hijack cell signaling pathways and can lead to such responses...requires successfully introducing receptors to target cells. Targeted and specific delivery becomes especially...ability of agonists to successfully reach target cells while resulting in minimal off target effects. For...

...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...Stop and smell the flowers - or rather, the lactate! The latest biosensor from Gary Yellen’s lab is LiLac...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...

...scFvs and T cell receptors and are expressed on T cells to make CAR T cells. The extracellular scFv portion...Bispecific T-cell engagers (BiTE®s), bind CD3 on T cells and a tumor-specific antigen, it brings T cells to a ...recognizes an antigen of interest while the intracellular T cell receptor portion of the CAR facilitates ...scFv part of the receptor can activate T cells to kill cancer cells. scFv’s small size also allows for their... display libraries screen antibody genes from B cells of immunized animals, but it's possible to avoid...also allows for screening scFv generated from the B cell antibody genes from diseased or vaccinated humans...produce scFvs in mammalian, yeast, plant, and insect cells, they are most often expressed from a plasmid in...

...Gordon Fishell New viral prep pAAV_BiSSTe10_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiCHATe27_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiVIPe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiLAMP5e3_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiPVe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiPVe3_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiSSTe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...

...co-expression among cellular markers You’ll also need to know if markers are co-expressed in your cells when designing...immunologists, allowing for the rapid analysis of cell populations within a heterogenous tissue type, such...methods, the identification and isolation of rare cell types. In recent years, full spectrum flow cytometry...detector to detect one fluorophore and therefore one cellular marker. However, emission spectra of certain fluorophores...Spectral flow allows for autofluorescence extraction Cells produce a low level of fluorescence called autofluorescence...your panel. If you have two highly overlapping cellular markers, it is important to pair them with two...similarity index of 0.87; therefore, two overlapping cellular markers should not be paired with these dyes, ...

...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...issues with tissue autofluorescence, cell heterogeneity, and/or cell position changes (To et al., 2016, ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP...cleavage by executioner caspases. Failure for cells to die or cells dying at the wrong time and/or place can...Apoptosis or “programmed cell death" plays a pivotal role in an array of biological processes including...including development, the immune system, and cell turnover. Apoptosis is a highly controlled process that ...sfCherry however did not initially fluoresce in HEK293 cells. To rectify this the Shu lab first improved the ...

...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...after transfection, and are quickly cleared from the cell via protein degradation pathways. This makes Cas9...

...Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Molecular Cell 9:789–798. https... Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent protein, YFP) is reunited by the interaction...directly visualize PPIs that regulate numerous cellular functions, including ubiquitinoylation, kinase...BiFC studies used the method to interrogate the subcellular localization of interactions between basic leucine...fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. The straightforward...involves three steps: fusion protein construction, cellular expression, and quantification (Kerppola, 2006...Fusion Proteins - Making the Right Connection. Cellular expression Whether BiFC constructs are expressed...

...mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced cells. Grant...in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org/10.1016/j.cell.2016.08.056 Find plasmids...targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from...the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general...pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression...for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD ...BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP...

... Alcaligenes eutrophus, Bartonella bacilliformis, Bordetella spp., Brucella spp., Caulobacter crescentus...Hyphomicrobium spp., Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium...rubrifaciens, E. stewartii, Escherichia coli, Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus...trifolii, Salmonella enteritidis, S. typhimurium, S. ordonez, Serratia marcescens, Shigella spp., Vibrio...ability to establish and maintain itself within host cells.  The plasmid has multiple ori that are functionally..., Azospirillum braziliense, Azotobacter spp., Bordetella spp., Caulobacter spp., Enterobacteriaceae, Gluconacetobacter...xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus...

...bottom of Tokyo Bay. When a weird, sulfurous smelling, black pellet was sent to the AbuGoot lab for testing...no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7-11...such as mouse embryonic stem cells and glioblastoma cells. PASTE Development and discovery The first ...observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s intrinsic machinery...turning potential into impact. In molecular and cellular biology, this happens through the development ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two...or DNA via a CRISPR mechanism. The weird black pellet's DNA yielded a new CRISPR effector protein they...