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We narrowed to 602 results for: ACE

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  1. Getting to Know Expansion Microscopy

    Type
    Blog Post
    Published
    June 20, 2023, 1:15 p.m.
    ...References and resources References Bertiaux E, Balestra AC, Bournonville L, et al (2021) Expansion microscopy...?  ExM provides a straightforward answer to the space problem. Instead of imaging smaller, what if we ...

  2. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    Published
    Sept. 8, 2016, 2:30 p.m.
    ...Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical biology 3.3 (2008): 142-155. PubMed PMID...from ensemble imaging to single-molecule imaging places a greater burden on the photon budget, which determines...

  3. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...H3 me1, me3, and ac – denoting mono-methylation, tri-methylation, and aceylation, respectively (these...a genomic region that has mono-methylated and acetylated histones, but not tri-methylated histones, functions...denote enhancers as follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll ...interests span machine learning, proteomics, and user interfaces for math.     Additional Resources Beyond the...

  4. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...Integration Using CRISPR-Associated Transposases. ACS Synth Biol 9:1998–2008. https://doi.org/10.1021/acssynbio...the cell. The edit in the chromosome can now take place. After editing, pTargetF can be cured from the cell... E. coli with the donor DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-...involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed...counterselection to yield pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF...serves as a counterselection marker and will be replaced by the sgRNA targeting the genomic site of interest... clearance. It is more convenient to update the spacer(20nt) on pEcgRNA plasmid as only two 24 nt oligos...

  5. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...Readout of Mechanical Forces in Biological Systems. ACS Sensors, 9(7), 3489–3495. https://doi.org/10.1021...

  6. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    Published
    Sept. 28, 2023, 1:15 p.m.
    ...Systems: The Rho-Family GTPase Signaling Toolbox. ACS Synthetic Biology, 11(1), 515–521. https://doi.org...and study a protein’s function at a very specific place and time. Future studies with these CIB1-Rabs in...

  7. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ...through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022 May 27;7(5):1336-1346. doi: 10.1021/acssensors...sensor Ace2N and additional GCaMP6 constructs: pAAV-CAG-dLight1.2 (AAV1) pGP-AAV-syn-FLEX-Ace2N-4AA-mNeon-ST...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....  Find B-gTEMP plasmids here!   Lu K, et al. Intracellular Heat Transfer and Thermal Property Revealed ...

  9. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    Published
    March 8, 2021, 2:41 p.m.
    ...Pillai-Kastoori L, Heaton S, Shiflett SD, Roberts AC, Solache A, Schutz-Geschwender AR (2019) Antibody...sample migrate through the stacking gel at the same pace and enter the resolving gel at the same time where...post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination. For example,...

  10. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ... Light-Inducible Gene Editing and Transcription. ACS Chem Biol 13:443–448 . https://doi.org/10.1021/acschembio...With their light-activated CRISPR/Cas9 effector (LACE) system, the Gersbach lab utilized a similar strategy...optimal fusion protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN...Far-red light, however, can penetrate 5 mm past the surface of skin and is less phototoxic. Using the FAST ...Activation of gene transcription Polstein et al., 2015 LACE CRY2 and CIB1 from A. thaliana dCas9 Activation ...

  11. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...or repression. The TCS light sensors described in ACS Synthetic Biology (2014) represent the newest versions...Photobiology: Optogenetics for E. coli. Schmidl, et al. ACS Synth Biol. 2014 Nov 21;3(11):820-31. Inncredible...compartment (e.g. the plasma membrane, the extracellular space, or the lysosomes) can take many paths. Understanding...that is quick, versatile, and robust. Dubbed the LACE system for light-activated CRISPR-Cas9 effector,...the CRISPR system. The ease and flexibility of the LACE technology makes this system widely accessible for...thereby creating a single-molecule reporter of intracellular processes. Multimerization via the SunTag system...combination of all 11 att pairs in series with unique spacer sequences (pMemoryArray) gives a DNA sequence capable...

  12. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...plasmids shipped as agar stabs. Iverson, et al. ACS Synth Bio. 2015. PubMed PMID: 26479688. RAS Pathway...modifiers like DNA methyltransferases or histone deacetylases. Unfortunately, these inhibitors are often non-selective... industry, the bioethanol industry, and the pharmaceutical industry, depend on the yeast, Saccharomyces..., including fermented beer and wine, fuels, pharmaceutical ingredients, and recombinant proteins. In order...

  13. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...1,000 active makerspaces around the world. Hackerspaces.org maintains a list of active spaces near you. ... can find a makerspace somewhere near you. The place to start looking for your local space is the Hackerspace...@derekja  Resources Hackerspace Website https://wiki.hackerspaces.org/Hackerspaces OpenWorm Project Website...manifested itself in the creation of Hackerspaces and Makerspaces back in 2007, which function as centres...with the OpenWorm Project One such space is the Victoria Makerspace in Canada, which I belong to. The ...  Funding Makerspaces One of the main challenges when starting a new project at a makerspace is finding...competition. In 2010, he founded makerspace.ca, Victoria’s first hackerspace which now offers members and ...

  14. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    Published
    Jan. 17, 2019, 1:38 p.m.
    ... take a look at the RSpace ELN.  The connected approach in action: how the RSpace ELN supports reproducibility...import them into RSpace. The kind of informal conversations about research that can take place in Slack normally...repositories. RSpace has been integrated with two of the five: Figshare and Dataverse, as well as the DSpace repository... reproduce an experiment documented in RSpace using the RSpace datasets deposited in Figshare or Dataverse...Rory is founder and CEO of Research Space, which provides the RSpace electronic lab notebook. Rory is based...contributed by Rory Macneil, founder of Research Space. There are many types of electronic lab notebooks...reproducibility RSpace was designed in response to requirements that emerged from intensive engagement with...

  15. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ...which makes them capable of diffusing in the extracellular space; they also have low immunogenicity and cellular...anterograde tracer and injected at several hundred sites across the entire mouse brain. Traced neurons expressed...it’s less likely to be sequestered in the extracellular space. In addition, rAAV2-retro’s altered amino...particles will diffuse long distances within the extracellular space along an axon, this does not seem to be the...brain tissue is injected with compounds that intracellularly spread away from the site of application and...suggest that there are several parallel mechanisms in place (for review see Maday et al., 2014). It is currently...Goshgarian, 2015). Nevertheless, the use of these tracers over the past few decades was essential to build...

  16. Build Your CRISPR Vocabulary

    Type
    Blog Post
    Published
    July 2, 2024, 1:15 p.m.
    ...with alternating spacers and repeats, was the original impetus for studying CRISPR. Spacers: Copies of foreign...sequences are called protospacers. Repeats: Bacterial DNA that appears between each spacer in the CRISPR array...sequence matches the guide sequence. PAM: The “protospacer-adjacent motif” present in the non-target strand ...the CRISPR array and include a spacer and a repeat sequence. The spacer sequence is the RNA guide, like...real mouthful of a phrase: Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity...composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed... into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins...

  17. Components of CRISPR/Cas9

    Type
    Blog Post
    Published
    Feb. 2, 2016, 5 p.m.
    ...provided that target is upstream (5') of a protospacer adjacent motif (PAM) Cas9 endonuclease binding to... and a 3-base pair sequence known as the Protospacer Adjacent Motif or PAM. In order for dsDNA to be cut...that each direct repeat, combined with its adjacent spacer, ultimately encodes a single crRNA. The direct...NNGRR(N)  Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) 5' TTTV AsCpf1 RR variant... and the unique stretches of DNA between them “spacers” (see figure below). After years of research, we...pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions, on the other hand, are the unique, foreign...tracrRNA forms the scaffold portion of a gRNA and the “spacer region” forms the target sequence.   Overview...

  18. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    Published
    Nov. 3, 2015, 3:30 p.m.
    ...question I have is, how did we get here in the first place? How CRISPR innovation affected software development...think we are currently at in the CRISPR software space. The CRISPR research community hasn't had a chance...review process has been challenged with many closely spaced CRISPR software submissions. With few prior publications... most value? This is only one of many decisions faced by those engaging in CRISPR design, but it is a ...significant hurdle made worse by an ever increasing marketplace. Since 2013 about 11 tools have been added every...given the lack of attention to it, it is an obvious place to focus further development toward. Quality control...main goals of the software and should be the first place you look to determine a tool’s suitability. Functions...

  19. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    Published
    May 8, 2018, 1 p.m.
    ...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region...excluding the PAM sequence. PAM Sequence - The Protospacer Adjacent Motif (PAM) sequence for the nuclease used...as determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing...and Sanger sequenced. ICE compares these sequence traces to give a detailed analysis of CRISPR editing. ...three tabs - Contributions, Indel Distribution and Traces - provides particular details about the indel profile...the reference sequence derived from the control trace file. On the plot, the green (edited sample) and...calls from the .ab1 file are also shown above each trace. The horizontal black underlined region represents...

  20. Healthcare Consulting: A Door to the Business of Life Sciences

    Type
    Blog Post
    Published
    Oct. 13, 2016, 2:30 p.m.
    ...Gairik Sachdeva. Healthcare consulting is a fast-paced field, requiring people who are willing to quickly... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance... commercial challenges or questions that clients face imminently. Consulting firms deploy case teams, ...all parts of a company to collaborate whenever it faces an important challenge. The types of projects mentioned...uncertainties and a rapidly evolving healthcare space. The consultants who enjoy their jobs most, get ...high levels of performance, and occasionally being faced with “shifting goal posts” as a part of a case, ...consulting can sometimes be challenging. The fast paced nature of projects often means working long hours...

  21. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...staining both cell surface and intracellular proteins in parallel, then stain the surface protein first, ... Figure 1: Cells expressing a particular surface receptor are stained with a fluorescent antibody...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...antibody thus amplifying the signal.       Intracellular protein targets, measured by fixing and permeabilizing...permeabilizing membranes For intracellular protein targets, users must first fix proteins within the cells...with formaldehyde first to freeze everything in place followed by alcohol permeabilization. Users should..., fix and permeabilize, and then stain the intracellular target. When using this approach, avoid fixation-sensitive...

  22. Clear and Accessible Writing

    Type
    Blog Post
    Published
    Feb. 15, 2024, 2:15 p.m.
    ...photo of an iodixanol gradient after ultracentrifugation. The interfaces between layers are muddled due to...the use of alt text to describe images in online spaces. Alt text is used by screen readers to provide ...from AAV Purification by Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader...for AAV Purification by Iodixanol Gradient Ultracentrifugation. The reader may be, for example, a graduate...between a gradient tube before and after ultracentrifugation so that a scientist can extract virus from...details of collecting the purified AAV–that the interfaces between layers are blurred after centrifugation...the syringe needle is inserted bevel-up at the interface between the 40% and 60% layers. Putting them all...

  23. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...antibodies for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently... control. This can involve cell surface labeling and/or intracellular labeling (or both). It is important...selectivity for cell surface labeling may become less selective if used in intracellular labeling because ...the intracellular compartment may contain cross-reacting antigens not present on the cell surface. The...best to determine this by testing them. For intracellular targets, we usually test 3 different fix/perms...helpful to try all three when looking to label an intracellular target.   Figure 1: HCT 116 WT and SYT1...optimizing fixation/permeabilization reagents for intracellular targets. Since knockout experiments can be ...

  24. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    Published
    March 14, 2017, 2:30 p.m.
    ...stage acquire genetic alterations and alter cell-surface antigen production to evade the immune system. ...APCs. Furthermore, tumor cells alter their cell-surface proteome to inhibit expression of co-stimulatory...histocompatibility complex (MHC) class I proteins on their surfaces to avoid recognition by CD8+ T cells. Existing...tumor formation and metastasis, block the cell-surface proteins that help escape immuno-surveillance, ...tumor microenvironment. Antibodies that block cell-surface tolerance-producing proteins are one of the most...penetration, inadequate concentration, mutation of cell surface targets by tumor cells, are a few of the reasons...producing chimeric antigen receptors (CARs) on their surfaces have been the most acclaimed form of immunotherapy...

  25. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ... the location of the ‘A’ relative to the protospacer adjacent motif (PAM). ABE7.10 can edit target 'A'... deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer. And if the target cytosine...A' if it lies in a small genomic window: at protospacer position 4-8 counting the ‘NGG’ PAM as 21-23 (...closer to the PAM, i.e., positioned at 8-10 in protospacer, ABE6.3 or ABE7.9 are better suited (Gaudelli...two Bsa1 sites to clone the single guide RNA (protospacer+scaffold) in a pol-III (OsU3) promoter driven...tRNA-gRNA (PTG) gene system to clone multiple protospacers which enables us to target more than one genomic...depending on the location of your target base in the protospacer sequence. If you do not find a suitable ‘NGG’...

  26. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    Published
    March 28, 2024, 1:15 p.m.
    ...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...linkage of intra- and extracellular environments. Found on nearly every cell in the human body, integrins are...eight beta subunits. Both subunits have short intracellular ends and large ectodomains containing ligand-binding...the cell membrane, physically connecting the extracellular environment and the cytoskeleton. Most integrins...affinity for ligands. When integrins interact with extracellular ligands, their ectodomains can extend and open...mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing agile responses...it is resisted by the ligand embedded in the extracellular environment. This further stabilizes the extended...

  27. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    Published
    March 24, 2016, 2:30 p.m.
    ...drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely spaced region allowed...approach to improve the efficiency of sequence replacement by HDR. Understanding Cas9 biophysics enhances...studies) to boost the efficiency of sequence replacement in vivo. We discovered that single stranded donor...Richardson et al., 2016.   dCas9 enables sequence replacement without cutting Our in vitro results also demonstrated...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...to tackle genetic diseases in which sequence replacement holds some kind of fitness advantage but error-prone...this technique supports gene disruption or gene replacement frequencies that are an order of magnitude greater...

  28. Viral Vectors 101: The AAV Retrograde Serotype

    Type
    Blog Post
    Published
    April 27, 2023, 1:15 p.m.
    ...information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular... either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...neurons that send information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons that...from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses...genetic information or tools, making them useful tracers, but finding a virus that works well for retrograde...that you understand this new tool, go forth and trace some neurons!  This post was originally written ...

  29. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    Published
    Sept. 7, 2017, 12:17 p.m.
    ...antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning rings, the remainder of...a 10 cm dish in 15- 20 minutes- a far more rapid pace than using cloning rings. A picture of a 60 mm dish... pipet tips (USA Scientific #1011-8410) is also placed in the hood. Finally, a sterile 2X DMEM solution...bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 cm dish containing...punched out, gently sucked up into the pipettor, placed into wells, and partially dislodged from the agarose...the plates that do not appear to be yielding well-spaced clones. For rapidly growing cells we sometimes ... TPP peelaway T-75 flasks to provide a greater surface area (the amount of agarose/DMEM should be proportionately...

  30. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ... a complementary DNA sequence, and (3) a protospacer-adjacent motif (PAM) on the opposite strand. Once... strand is edited by base editors! PAM Protospacer-Adjacent Motif A short sequence that must be present...an —NH2 (amine) group from the target base and replaced it with a double-bonded oxygen. This simple enzymatic...Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop...that preferential C → G editing was possible, the race was on to develop more efficient and precise editors...activation domain (SAD), which recruits histone acetyltransferase to open chromatin, improved editing efficiencies... editing, an alternative editing strategy that replaces a target region with a newly synthesized, templated...

  31. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    Published
    Aug. 15, 2019, 12:30 p.m.
    ...mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile technique...modifications to pDS132 (Philippe et al., 2004): Replaced the sacB cassette with one of three toxins from... a broad array of pathogenic Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible...the Salis group) (Espah Borujeni et al., 2014). Replaced the pDS132 MCS with a versatile, synthetic MCS... “pTOX” in a broad group of Gram-negative Enterobacteriaceae including in E. coli O157:H7, Shigella flexneri... point mutations. I think it will always have a place in every bacterial geneticist’s toolbox. Depending...negative selection systems for unmodified Enterobacteriaceae." Nucleic acids research 43.13 (2015): e83...

  32. Hot Plasmids: Summer 2024

    Type
    Blog Post
    Published
    Aug. 27, 2024, 1:15 p.m.
    ...assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1)....determination. A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm...software (green circles), magnetic bead (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of ...other targets. The modular system of linkers and spacers could enable many future variations using nanobodies...Cryo-EM samples can be damaged at the air-water interface (AWI) during the freezing process, resulting in...When proteins (blue) interact with the air-water interface (AWI) before vitrification, they can be completely... Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM ...

  33. Learning Technical Skills at a Lab Central Workshop

    Type
    Blog Post
    Published
    Feb. 20, 2018, 2:30 p.m.
    ...new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared by ...and making space for the techniques ourselves. As you probably have limited time and space in your own...by numerous emerging biotech companies. The space was modern with clean bold colors of orange and white...like workshops at Lab Central. Because of the fast pace of work in the Addgene lab, we don’t often get the...opportunity to explore new techniques in our own space. Workshops such as this give us a chance to learn...

  34. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ...many histidine residues to include.  Placement As far as placement goes, there is no cut or dried answer...frequency amino acid, proteins containing two or more adjacent histidines are present in most production systems...polyhistidine tag plasmids here! Where should I place the tag and how many do I need? When designing a...ions once the protein is folded. Sometimes a tag placed on one end will be blocked during protein folding...should try tagging the opposite end.  If neither placement works, consider purifying under denaturing conditions...terminus is preferable over the other however, tag placement can affect protein function. B, The string of ...

  35. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    Published
    Jan. 19, 2021, 2:15 p.m.
    ... B cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes...microbes, or to proteins expressed on the surface of cells that have been infected with a microbe, to trigger...the amount of protein present. The reaction takes place in a 96-well plate, where primary and secondary ...Flow cytometry is used to detect proteins on the surface of or inside whole cells. Antibodies - typically...cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be permeabilized to allow... allow antibodies to bind to intracellular proteins. The fluorescence signal is detected by the flow cytometer... activate cellular pathways by binding to cell surface receptors. These applications are typically used...

  36. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...receptors expressed on the cell surface. Thus, a screen can be used to target surface proteins to determine which...known to cause the up-regulation of an important surface protein (e.g. PD-L1 on a cancer cell). In this ...signaling molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different...transferred cells is followed. A benefit of working with surface proteins is also that such proteins are easily ...further be used to interfere with the identified surface proteins, and such antibodies could thus be interesting...all differentially expressed genes that code for surface proteins. One of the first challenges with a custom...the information that you have input in the web interface, making it convenient for you to later identify...

  37. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    Published
    July 19, 2016, 2:30 p.m.
    ...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,... to get your fluorophore delivered to the right place? FRET experiments require targeting the donor and...specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET acceptors,... site when used in conjunction with Cy dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright,...proteins (such as GPCRs, ion channels, or cell surface receptors) or with experiments on permeabilized...Biarsenical labeling reagents If you want to label intracellular proteins covalently with FRET acceptors, then...and the acceptor fluorophore binding sites are adjacent to each other on the same protein and a negative...

  38. Rabies and Neuronal Tracing

    Type
    Blog Post
    Published
    May 29, 2018, 1:51 p.m.
    ...transsynaptic neuronal tracer into a monosynaptic (first order) neuronal tracer For RABV to crawl through...neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability to efficiently enter cells...However, they generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically...cells through their interactions with target cell surface receptors (Callaway 2008). Based on this, cell ... RABV expressing the viral protein EnvA on its surface will only infect Cre-positive, TVA-expressing cells...taken up by the presynaptic nerve terminals of adjacent neurons (i.e., it is taken up by projection neurons...

  39. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ...better access to intracellular targets. If your target is already on the extracellular side of the membrane...proteins in place, while allowing antibodies into the cell where they could bind to intracellular epitopes... Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating and ...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...is suitable for antibodies that bind to the extracellular portion of a protein. B) Permeabilizing with...

  40. Congratulations, Deck The Lab winners!

    Type
    Blog Post
    Published
    Jan. 14, 2021, 1:30 p.m.
    ...Lab: Classic Edition Winners In a tie for first place, the Vestibular Lab decorated not just their lab...@druryhannah) The Tarling-Vallim Lab’s first place-winning entry doubled as their lab’s decoration ...Lab (@TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual...Deck the Lab: Home Edition Winners Winning first place in the home edition, the MicrobiGals created glow...Microbigals (@MicrobiGals) December 23, 2020   In second place, Dr. Anne Madden created a microbe advent tree displaying...Madden (@AnneAMadden) December 23, 2020   In third place is Matthijs Dorst’s Deck the Lab Nativity scene ...

  41. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    Published
    Aug. 20, 2020, 2:30 p.m.
    ...the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide...modifications at DNA targets adjacent to NGG PAM sequences when used in place of wild-type SpCas9. The remaining...enzymes require a single base protospacer flanking sequence (PFS) adjacent to the target, but many do not... REPAIR (RNA Editing for Programmable A to I Replacement) system fuses Cas13b to RNA deaminase ADAR2 to...

  42. Transferable Skills Guide: Creativity

    Type
    Blog Post
    Published
    Jan. 18, 2022, 2:15 p.m.
    ...Penguin Publishing Group. Day, F. (2019). Embrace your weird face your fears and unleash creativity. Gallery...focus. Find yourself a block of time and a quiet space where you can address the issue head on. Prepare...can come any time. Meetings shouldn’t be the only place people share ideas. Ideas had over lunch are just.... Identify your enemies In Felicia Day’s book Embrace your Weird, Day identifies nine enemies that stand...backordered for months, we have to have plans in place to be able to continue our experiments. Thinking...team has used these techniques to optimize our lab space, our protocols and even our Lab Inventory Management...

  43. Tips for Writing a Good Cover Letter

    Type
    Blog Post
    Published
    May 21, 2024, 1:30 p.m.
    ...paragraph is the place to introduce yourself and your background. It’s a great place to state why you’...What is a cover letter for? A cover letter is a place to provide helpful context about your candidacy,...candidacy, beyond what is on your resume. It is a space for you to explain how your skills and experience match...candidate!  Additional uses The cover letter is a great place to proactively address questions about your application...you haven't talked about it yet, it's also a good place to state why you are interested in working for this...applicants. In short, while a cover letter can’t replace skills and experience, it can help the hiring manager...

  44. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    Published
    April 11, 2024, 1:15 p.m.
    ...Bordetella spp., Caulobacter spp., Enterobacteriaceae, Gluconacetobacter xylinus, Haemophilus influenzae,...Gram-negative and Gram-positive  Acinetobacter calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces...carotovora, E. chrysanthemi, Escherichia coli, Gluconacetobacter xylinus, Gluconobacter spp., Hyphomicrobium...pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens, A. salmonicida, Agrobacterium...campestris pv. campestris, X. campestris pv. malvacearum, Zymomonas mobilis pBBR1 based pBBR1 ...Caulobacter crescentus, Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans, Pseudomonas...

  45. IDP and your PI: A Roadmap for Career Planning and Personal Development

    Type
    Blog Post
    Published
    July 21, 2015, 2:30 p.m.
    ... Angela DePace has put personal development at the center of her lab (pictured above). DePace holds yearly...through thoughtful planning and honest feedback DePace’s yearly planning meetings revolve around two simple...tone of the meeting is almost more important. As DePace says, science is full of “terrible perfectionists...have tangible benefits for both PI and trainee. DePace and her lab members have noticed that the meetings... Park J, Bragdon MD, Lopez Rivera F, Biette KM, DePace AH. Mol Cell. 2015 Jun 4;58(5):718-21. doi: 10.1016...

  46. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ... in the extracellular domain which activates the native signaling cascade of the intracellular part of...ligands (PORTLs). OptoXRs are chimeras of the extracellular domain of endogenous light-sensitive receptors...receptors, like mammalian rhodopsin, and the intracellular domain of an endogenous GPCR, such as adrenergic...signaling pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be...state.      Figure 4: Example of an intracellular signaling pathway activated by an optogenetic...., & Cui, B. (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology,...

  47. Finding Your Science Policy Path

    Type
    Blog Post
    Published
    Sept. 22, 2020, 1:15 p.m.
    ...rich and diverse workforce. Out of all the career spaces I explored for my post-PhD life, the science policy...science policy. Luckily, there are many mechanisms in place to ensure your effective networking---and they’re...pandemic has foisted upon us. Action Items:  A simple place to look for science policy folks is on Twitter- ...! Someone from your alumni community is a great place to start. What is science policy? THE IMPOSSIBLE... policy is so diverse, you can do it in so many places-- not just the government. When you talk to folks...technologies accessible to patients. And what better place to do that than at the nation’s largest integrated...to chat with someone working in a science policy space you’re interested in. Many fellowship programs can...

  48. Reagent Repositories Are Speeding up Science During the Pandemic

    Type
    Blog Post
    Published
    Aug. 4, 2020, 1:15 p.m.
    ... entered human cells via hACE2, could not enter cells expressing murine ACE2 (Subbarao et al., 2006). ...SARS-CoV-2. The resurgence in popularity of the hACE2 transgenic mouse strain, for example, is sustained...the University of Iowa (McCray et al., 2006), the hACE2 transgenic mice were designed after it was discovered...Repositories around the world facilitate the rapid pace of SARS-CoV-2 research by removing logistic hurdles...Look DC, Perlman S (2006) Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome...

  49. Antibodies 101: Isotypes

    Type
    Blog Post
    Published
    Oct. 19, 2021, 1:15 p.m.
    ... that helps attack extracellular pathogens.  Transportation of antibodies to places they can’t reach on...valency of 10 antigens. An extra constant domain replaces the hinge region in IgM antibodies. IgM has no.... IgD While IgD is coexpressed with IgM on the surface of most immature B cells, its function is unknown.... They are found bound to IgE receptors on the surface of basophils and mast cells, two types of white... the intervening DNA sequence is deleted, which places the variable region next to a different constant...

  50. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    Published
    Nov. 19, 2019, 2:08 p.m.
    ...target proteins. The surface-exposed loops of nanobodies seemed like a good place to put AsLOV2 because...inserted AsLOV2 into conserved sites in all eight surface-exposed loops of an mCherry nanobody. Only insertion...-dependent shift in mCherry fluorescent to the surface of the bead, suggesting that OptoNBs still bound...bound their target protein even when stuck to the surface of a bead.  Using OptoNBs to control cell signaling...With darkness, SOS binds Ras and initiates the extracellular signaling-regulated kinase (ERK) signaling cascade...
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