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We narrowed to 593 results for: cel.2;

Showing: 81 - 120 of 593 results

  1. CRISPR Plasmids - Base Edit

    Type
    Collection
    ...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme... inosine, which is treated like guanosine by the cell, creating an A to G (or T to C) change. Adenine ...

  2. Reagent Repositories Are Speeding up Science During the Pandemic

    Type
    Blog Post
    ...Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV-2 were developed for ...different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in this study came from...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...that science is truly a global endeavor. Case study 2: Plasmids Another example of reagents that have broad...applications but have proven useful to the study of SARS-CoV-2 are the lentiviral production and packaging vectors...

  3. CRISPR Plasmids - Drosophila

    Type
    Collection
    ...plasmid Bullock and Port 49409 pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair ...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...

  4. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...that cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...in tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted ... overall (GFP+ cells that also stained for a neuronal marker). Check out tables 1 and 2 below for a summary...high dose to maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e...require the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in...

  5. New and Upcoming Viral Vectors - May 2020

    Type
    Blog Post
    ...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...

  6. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...provide a 2-10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...

  7. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number of cell types at different...Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...

  8. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ...Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020. (B) Mapping...designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface. The Bloom lab used ...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...

  9. Gendered Innovations: Why Does Sex of the Cell Matter?

    Type
    Blog Post
    ...state-of-the-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how...never considered the sex of their cells. For one thing, cells and cell lines degenerate over time—and sex... effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to ...therapeutic capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—which...sexes in basic research is excellent design), but they used all female stem cells—this was an unconscious... stem cells. Nor did they detect important differences in function between male and female cells. The ...experiment. In the case of stem cells, these factors may include cell type, the disease being treated,...

  10. Antibody Production

    Type
    Collection
    ...for shipping stability by incubating at 37 °C for 2 weeks, freezing at -80 °C, thawing on ice, and then...mammalian expression vectors, human embryonic kidney cells are transfected with the plasmid(s) encoding the.... This buffer is not compatible for use in live cells and will interfere with conjugation. For applications...antigen is first transiently expressed in mammalian cells. To learn which specific QC experiments were performed...email [email protected] . Immunocytochemistry (ICC) Cells are fixed and labeled with the antibody and a species-specific...Western Blotting (WB) Proteins are extracted from cells, separated by SDS-PAGE, transferred to a membrane...

  11. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ...interest in the cells you are studying. Read our SPARC blog post for more details. Figure 2: Dense (c... rising intracellular calcium levels within the cell. When levels of Ca2+ are high in the cell, calcineurin...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The... of a genetically-defined subset of cells. By labeling fewer cells in a population, it’s easier to visualize...lengths, the density of cells that are labeled can be altered to dense (45% of cells labeled), intermediate...or aging cell populations. Figure 3: Micrographs of wild type (top panels) and cells with high...barcodes (iBARs) in their loop region. After infecting cells with sgRNAiBAR library containing lentiviral vectors...

  12. Luciferase Plasmid Collection

    Type
    Collection
    ...transfection Feng Zhang 21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase...and Venus fluorophore to Troponin C Ca 2+ binding domain. Luminopsins : Luciferase-opsin fusion proteins...Expression of firefly luciferase from a cell-free mammalian cell lysate Marcel Bruchez 108542 pLenti-EF1a-Luciferase-IRES-Blast-WPRE...not secreted, so cells must be lysed or the substrate must be delivered intracellularly to measure luminescence...expression of luciferase in the cytoplasm of mammalian cells Peter Cockerill 212936 pGL3 Basic Vector Firefly... viral infection efficiency, to study changes in cell physiology, or as a reference level of expression...Expression of Gaussia luciferase; GFP is expressed if cells are infected with virus Christopher Buck 98580 pLX304...

  13. Deisseroth INTRSECT Collection

    Type
    Collection
    ...dual-transgenic recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing...delivering additional recombinases 10–14 . Figure 2: INTRSECT works by inserting short, intronic sequences...well-defined cellular sub-populations has generally been limited to single-component cellular definitions...to enable three-feature cell targeting. C) Example of viral targeting of cells expressing Cre AND Flp,...parameters. Figure 1: Examples of intersectional cell population definitions using recombinases. A) Cre...adeno-associated virus (AAV)-borne payloads to be expressed in cells based on a doubly-specified combination of genetic...multiple recombinase-expression strategies to define cellular sub-populations of interest, including dual-transgenic...

  14. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

    Type
    Blog Post
    ...Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 184:2372-2383...months, we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts...They were able to tweak this assay to study SARS-CoV-2 spike variants. “We created a series of spike expressing...relative ability to neutralize.”   Figure 2: Sera used in the study came from individuals who ...This sera was used in the high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was...viruses are quite genetically distinct from SARS-CoV-2 and its variants. Yet, the B.1.351 variant, which ...effectiveness of current vaccines against SARS-CoV-2 variants, this study was the first to compare so many...

  15. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate research and to help ...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

  16. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    ...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T cells in our first study...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

  17. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ...this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen... hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer... tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving..., and the stem cell lines we’ve made using them are available as part of the Allen Cell Collection at ...

  18. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...change the media (add 2-3 mL of fresh media once a week), and do NOT harvest the cells before at least 10...titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 cells and produce more rAdV ...infected cells show this “cytopathic effect.” Amplification can be repeated multiple times (2-4 rounds...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result in a 10...

  19. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1...spike protein’s role in infecting cell lines without using intact SARS-CoV-2 virus. Scientists have been using...Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi....production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid... pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details...

  20. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

  21. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...

  22. Distribution to Industry

    Type
    Collection
    ...and HaloTag Fusions from Promega COVID-19 SARS-COV-2, ACE2, and CRISPR tools Fluorescent Proteins from ...plasmids for detecting GPCR activity in a variety of cell types. Protein Purification Optimization Kit Using...

  23. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    ...: 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ...Make sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay...contaminants. Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine...’s best to split cells before the plates are more than 80-85% confluent because cells like their space...lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split very...numbers! When starting off with a new cell line, grow the cells, aliquot them into a number of vials, ...

  24. The Challenges of Cell Culture

    Type
    Blog Post
    ... York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University of New York...quality work in cell culture. The problems with cell lines Simply put, the majority of cell line problems...HeLa cells, the first immortalized human cell line. He proved this by examining whether the cell lines...Cancer Cells, Marker-independent identification of glioma-initiating cells. The International Cell Line ...working cell bank has been made by a research institution (an excellent practice), or maybe cells have been...Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture... Outreach Scientist Nick Amiss. Cell culture in the present day Cell culture is a widespread tool used...

  25. Typing CRISPR Systems

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    Blog Post
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...). Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing. Cell & Bioscience...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...

  26. Tetracycline Inducible Expression

    Type
    Collection
    ...hSyn promoter rtTA3 Wei Xu 26803 pEnt L1L3 EF1a-tTA-2 Gateway entry vector to express tTA from EF1α promoter...expression and improved dynamic range . Hum Gene Ther, 22 (2), 166–176. https://doi.org/10.1089/hum.2010.099 (Link...Plasmids Collection Cre-lox Plasmids Collection Stem Cell Research Plasmids Collection Tetracycline Off Tetracycline...expression of the transactivator proteins in mammalian cells or other species by codon optimization of their ...gene of interest from two separate vectors. Stable cell lines or transgenic animals can be made to achieve... and ensures that all transfected or transduced cells express both components in the same relative ratio...derivatives can be found in Krueger et al., 2004. Some cell culture sera may contain tetracycline or its derivatives...

  27. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to...construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but...Deliver your construct to your target cells. After recombination, exon 2 of the targeted gene will be removed... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...

  28. Brain Initiative Collection

    Type
    Collection
    ...Gordon Fishell 83899-AAV1 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAV9 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAVrg pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...real-time interrogation and monitoring of brain cells and circuits during behavior. Long desired by researchers...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Human...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Cre-dependent...genetically encoded dopamine sensor RdLight1 in mammalian cells. Lin Tian 125708-AAV9 pAAV-hSynapsin1-RdLight1 Expresses...

  29. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    ...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...multiple plasmids in one cell, it might be that you can't stably maintain them in the cell. This blog post features...

  30. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...but these cells are prone to mutations and will have more unintended changes to their genomes. 2) Designing...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....

  31. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...   Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with ... a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially if you’re doing...(Figure 2) and I was surprised at how variable Cas9-expression was across the different cells (Figure ...Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn’t know what...knockouts and knockins in human somatic cells. Nature Protocols 2007; 2(11): 2734-46. PubMed PMID: 18007609...despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you really...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...

  32. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic... multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...

  33. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,... and keep the cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend...

  34. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

  35. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    ...electrocompetent cells is minimal (picogram range). It can be extracted from small bacterial cultures (i.e. 2-3 ml...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...

  36. Hot Plasmids - October 2022

    Type
    Blog Post
    ...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...challenges in 2-photon in-vivo imaging of voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  ...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec;5(12):3610-6.(1985). doi: 10.1128/mcb.5.12.3610...

  37. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...α2,3-/α2,6 N-linked sialic acids1 AAV receptor (AAVR)2   AAV2 Heparan sulfate proteoglycan (HSPG)3 ...3 AAV receptor (AAVR; not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5...AAV5 2,3-N-linked sialic acid4 AAV receptor (AAVR)2 Platelet-derived growth factor receptor (PDGFR...Heparan sulfate proteoglycan (HSPG)3 AAV receptor (AAVR)2 Epidermal growth factor receptor (EGFR)3 AAV8... AAV8 Laminin receptor (LR)3 AAV receptor (AAVR)2 Carboxypeptidase D (CPD), also called AAVR25   ... Terminal N-linked galactose3 AAV receptor (AAVR)2 Laminin receptor (LR)3 Putative integrin3 ... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...

  38. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...

  39. CRISPR Plasmids - Empty gRNA Vectors

    Type
    Collection
    ...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...BclI-SwaI none S. pyogenes URA3 Wyrick pCRISPomyces-2 61737 Bacteria BbsI yes, cut S. pyogenes Apramycin... S. pyogenes Zhang gRNA Cloning Vector Bbs I ver. 2 85586 Mammalian BbsI None S. pyogenes H Fujii pX601...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...Alternatively, gRNA-only plasmids can be used in cells or organisms in which Cas9 has been integrated into...

  40. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates,...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express...
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