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  1. Hot Plasmids Spring 2024

    Type
    Blog Post
    Published
    May 23, 2024, 1:15 p.m.
    ...our Hot Plasmids articles.  Here's what you'll find in this post: Multiplexed perturbation and decoding... detection efficiency over the original CROPseq. Find CROPseq-multi plasmids here Walton, R.T., Qin, ... editing and found the protein La (a small RNA-binding exonuclease protection factor) promotes editing...improved efficiency is at least in part due to La binding and stabilizing the 3’ end of the pegRNA, which...additional potential improvements to the editor. Find PE7 plasmids here!     Figure 2: PE7 improves...Improving prime editing with an endogenous small RNA-binding protein. Nature. 2024 Apr 3. doi: https://doi.org...their paper for details on the original antibodies. Find Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et...

  2. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...(PAM) on the opposite strand. Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of... have an activity window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...strand of DNA for Cas9 to bind.   R-loop The structure formed by Cas9 binding to DNA and separating the...specificity and the R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary...at certain positions within the editing activity window (Kurt et al., 2021). They hypothesized that the...components: the DNA repair protein rXRCC1, or the DNA binding and lyase domain of DNA polymerase β (PB) (Chen...

  3. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    Published
    Feb. 28, 2017, 3:30 p.m.
    ...on Asddgene.org Find Fluorescent Protein Plasmids Find Fluorescent Protein Kits Find Empty Backbones ...hairpin binding domains produce blue, green, or red fluorescence, depending on the hairpin binding domain...researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach...in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed ... when a. no gRNA was present or b. when a gRNA binding a non-mammalian sequence (GAL4) was supplied. ..., GFP, and RFP fused to the respective hairpin binding domain. Pairs of identical hairpin sequences specify...hairpin combination bringing the total number of individual colors to 6. A gRNA with all three hairpins generates...

  4. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage indicators (GEVIs)...blue-light-sensitive channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al...functional. Exposure to red light induces PhyBΔ and PIF6APB binding, bringing the two halves of Cre together...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like...therapeutic potential for the split base editors. Find the split base editors at Addgene. Levy et al., ...fluorescent optics. Existing red fluorescent voltage indicators, however, fall short compared to GFP-based sensors...linker between them. The Ace-mScarlet voltage indicator works like many FRET-opsin GEVIs. With an increase...

  5. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find more new CRISPR tools for your research! This time...the paper in Cellular and Molecular Bioengineering Find the optogenetic repressor plasmids Cytosine base...editing The original cytosine base editor, BE3, induces a low frequency of genome-wide off-target DNA editing...identified and validated CBEs with reduced Cas9-independent deamination. They generated these editors using...this study have ~10-100-fold lower average Cas9-independent off-target DNA editing, ~5–50-fold lower levels...BE4max.  Read the paper in Nature Biotechnology Find the base editor plasmids CRISPRi in Caulobacter...

  6. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...to be validated for high on-target binding and low off-target binding. It’s also possible that HIV-1 may...immunodeficiency syndrome (AIDS) in untreated individuals. Symptoms of AIDS include rapid weight loss and...opportunistic infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate...targeting of the enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...CRISPR. CD4+ T-cells were isolated from healthy individuals, expanded, and infected with HIV-1. For two HIV...infected patients. Although this is an exciting finding, it’s important to note that these HIV-1 patients...

  7. Antibodies 101: Epitope Availability

    Type
    Blog Post
    Published
    Jan. 11, 2022, 2 p.m.
    ...protein, the antibody can't bind to it. As proteins change configurations or binding partners, the epitopes...instance, if you cannot find an antibody validated for Western blots, but find one validated for ELISAs...antibodies are well-organized, documented, and easy to find (though we might need to see it to believe it.) ...) As you browse the collection, you'll likely find some proteins of interest for which you have several...simple: epitope availability. An antibody can only bind to a target, or epitope, that it can access. If ...epitopes available for binding change as well. If this change hides the epitope that the antibodies used to ...antibodies, so it's good to keep epitope availability in mind whenever you're working with antibodies.) Conformation...

  8. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...intracellular staining where binding of large immune complexes may be hindered. Indirect staining can be beneficial...conjugated to a fluorochrome. In indirect staining a primary antibody binds to a target and a fluorochrome-conjugated...secondary antibody binds to the primary antibody. Direct staining is quicker than indirect staining and eliminates...viability controls such as DNA binding dyes and amine reactive dyes. DNA binding dyes, such as propidium iodide...the challenges of fluorescent protein fusions by binding to the protein in its native state.  Antibodies-based...Antibodies-based flow cytometry can use both direct and indirect staining methods. In direct staining, the primary...levels are low as multiple secondary antibodies can bind to a primary antibody thus amplifying the signal...

  9. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    Published
    Dec. 10, 2019, 1:58 p.m.
    ...fused to an RNA binding motif, and a RNA-binding protein-bEFP fusion. Thus, the bEFPs bind the UAS via the...Transcription from σ54 promoters requires binding of bacterial enhancer binding proteins (bEBPs) at a sequence upstream...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like...genetic screens in cells with few genetic tools. Find the mouse CRISPR knockout library at Addgene. Henriksson...plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol...strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol...understanding and treatment of cardiovascular disease. Find the LipoGlo plasmids at Addgene! Lawler et al, QJM...

  10. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ...chance of generating indels with base editors, keep in mind that the generation of indel sometimes may also...Addgene.org Find all CRISPR plasmids and resources at Addgene Find CRISPR base editors Find CRISPR plasmids...reducing the likelihood of detrimental indel formation. After binding with DNA, the CBE deaminates a target...heard about CRISPR, one question probably comes to mind: can CRISPR be applied to fix the defective base...the genome became achievable in 2016 when two independent groups (Komor et al., 2016 and Nishida et al....base editor would restore antibiotic resistance. To find such a protein, they created a mutant library of...can edit target 'A' if it lies in a small genomic window: at protospacer position 4-8 counting the ‘NGG’...

  11. Developing a cold tolerant E. coli using specialized chaperones

    Type
    Blog Post
    Published
    Jan. 28, 2020, 2:35 p.m.
    ... for the 2019 All-India iGEM Meet-up, which served as a platform for all the Indian iGEM teams to interact...the Addgene blog Find our synthetic biology blog posts Resources on Addgene.org Find plasmids for your... the bitter cold.  Since our objective is to induce tolerance towards cold, we decided to focus on ...The importance of psychrophilic enzymes From an industrial perspective, the enzymes obtained from psychrophiles...diverse array of processes. The food processing industry, for instance, employs psychrophilic enzymes for...extremely useful benefits for any food processing industry as it can potentially help minimize the incidental...spoilage and improve the productive efficiency.  Finding recombinant psychrophilic enzymes for the host ...

  12. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...transfer vector with an inducible promoter (see below) should be used. Inducible lentiviral transfer vectors...containing both your transgene and tTA/rtTA (pINDUCER20, pINDUCER21) have been developed, but these vectors...Nature found a way to re-create a functional virus. Indeed, by testing several vectors and packaging cell ...your gene of interest either constitutively or inducibly. If your gene of interest is toxic or needs to...vectors The most widely used inducible lentiviral vector system is the tetracycline (Tet)-regulated system...transactivator (tTA, also expressed by your transfer vector) binds to the TRE and activates the expression of your ...system, tTA has been modified (and renamed rtTA) to bind TRE only in presence of Dox. In this case, your ...

  13. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    Published
    Nov. 24, 2015, 3:30 p.m.
    ...Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP. Once...love GFP, and we’re always excited when depositors find new ways to make this workhorse protein even more...Once Tang and Cepko found a description of GFP-binding nanobodies, the project really took off. Unlike... are small and stable inside cells. Nanobodies binding GFP were designed in 2009. Subsequent work showed...proteins, and these fusions retained the ability to bind GFP. The idea of using GFP as a scaffold suddenly... GBPa-VP16 (activation domain); GBPb-GAL4 (DNA-binding domain); and a UAS-driven luciferase reporter construct...fluorescent proteins dsRed, mCherry, and TdTomato do not induce transcription. Thus, T-DDOGs can be used in combination...

  14. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    Published
    June 3, 2021, 1:15 p.m.
    ... a GCN4 binding scFv that’s fused to GFP. When the multiple copies of the scFv-GFP fusion bind the SunTag...cell. When a CAR has an scFv that binds cancer-related antigens, binding of the scFv part of the receptor...the smallest unit of an antibody molecule that can bind antigen. While they may seem unfamiliar, scFvs are...favors proper folding and formation of the antigen-binding site while also minimizing oligomerization of the... can generate functional scFvs; however, some individual scFvs performing better in one configuration ...antigen to select for scFvs with high affinity binding. Bound phage are then proliferated by infecting...screened with in vitro assays to select for strong binders. Then the sequences for the variable domains of...

  15. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

    Type
    Blog Post
    Published
    Aug. 24, 2021, 1:15 p.m.
    ...from Boguszewska et al., 2019.   Indirect ELISA The indirect ELISA is very similar to a direct ELISA...sample adheres to the well (either directly or by binding to an antibody that is coating the bottom of the.... These are sandwich, competitive, direct, and indirect. Sandwich and competitive ELISA use antibodies...is bound directly to the wells in a direct and indirect ELISA.   Sandwich ELISA Most commercially available...add your sample to the wells, and the antibodies bind to your protein of interest.  The next step is to...biotin-conjugated, streptavidin (which has a high affinity for binding biotin), is conjugated to the enzyme horseradish...protein — known as a reference antigen — that can also bind to that antibody. Then, the protein-antigen mixture...

  16. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    Published
    April 25, 2023, 1:15 p.m.
    ...requires indirect staining of both targets in your specimen. You scour the literature and find an ideal...using the indirect method since a secondary antibody reactive to a certain isotype will bind to all members...antibody to bind to the anti-Protein A antibody and an anti-mouse IgG2a secondary antibody to bind to the ... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...the same isotype then the secondary antibody will bind to both and you will not be able to distinguish ...the same isotype can not be used for multiplexed indirect staining methods since the secondary antibody ...primary antibodies and the targets will be indistinguishable (top panel). If one of the antibodies is converted...

  17. In Vivo Biotinylation of Bacterial Fusion Proteins

    Type
    Blog Post
    Published
    Jan. 25, 2018, 2:09 p.m.
    ...ligand binding site. A selective approach entails the use of the enzyme biotin ligase, which binds to a ...Check out other Hot Plasmids Find Additional Posts on Mol Bio Techniques Find Additional Posts on Plasmid... Rex's lab. A common strategy to determine the binding kinetics of a purified protein involves immobilization...bound ligand (after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST)... GST moiety, which forms dimers, may influence binding kinetics of the target ligand. Another important..., protein immobilization through biotin-avidin binding could provide a true measurement of high affinity...interactions. Avidin and its derivatives have four biotin-binding sites per molecule so, theoretically, saturated...

  18. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    Published
    Sept. 18, 2018, 1:33 p.m.
    ...of cobalamin-binding riboswitches that bind to cobalamin-fused fluorescent dyes; binding of the cobalamin...development of these probes, fluorophore binding aptamers and MS2-binding fluorescent proteins were the state...number of fluorophores. You can find plasmids containing the cobalamin binding riboswitches used in the Riboglow...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like...consists of two domains, LOV2 and Zdk1, where Zdk1 binds LOV2 with high affinity in the dark but dissociate...halves (Fig. 1): one containing the microtubule binding domain and the second containing the growth promoting... could be stopped in areas with blue light - a finding which opens novel ways in manipulating and studying...

  19. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...The field of induced pluripotent stem cells (iPSCs) has been around for 10 years. In that time, scientists...readers choose a strategy suited to their research. Find Plasmids for Stem Cell Research Delivery Method... iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses address some of these concerns, but...reprogramming. This can be overcome by using a Tet-inducible virus to allow for controlled expression of reprogramming...links at the end of the post for a list of Tet-inducible reprogramming vectors available from Addgene. ...loxP sites. While there is only a small loxP scar behind at the site of excision, this approach is still...of cell types and replicates in the cytoplasm independent of the cell cycle. A challenge of using Sendai...

  20. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    Published
    Nov. 29, 2022, 2:15 p.m.
    ...make sense in any kind of biological framework, suggested to them that their findings may be the result...lines, looking to uncover genetic dependencies of individual tumors, which can lead to new cancer targets ...ancestry of the cell lines (European, East Asian, Indigenous American, Africa, or South Asian) used in the...significant statistical limitations, and expected to find only a few, very strong associations.   Unexpected...Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry associated dependencies...of a true correlation.   It turned out there was indeed a bias, and it was coming through an unexpected...source: the CRISPR guides, short pieces of RNA that bind to a cell’s DNA, telling the Cas machinery where...

  21. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing vectors at Addgene! Multiplexing...expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet),...), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid...plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more ...PMID: 25337876. PubMed Central PMCID: PMC4270925. Find plasmids from this paper at Addgene. 2. Kabadi,...PMID: 25122746. PubMed Central PMCID: PMC4231726. Find plasmids from this paper at Addgene. 3. Sakuma,...PMID: 24954249. PubMed Central PMCID: PMC4066266. Find plasmids from this paper at Addgene. 4. Albers,...

  22. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ... many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous...process! As NHEJ is ineffective in bacteria, CRISPR-induced double stranded breaks (DSB) are lethal. Addgene...persists, Cas9 will cleave the gene of interest, inducing a lethal DSB.     This system is distinct from...expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet),...), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid...plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more ...Enterobacteriaceae, without the need for modification. This finding suggests that the system may be functional in most...

  23. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    Published
    March 30, 2017, 2:30 p.m.
    ...This post was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta. If you...in real time PCR, each reaction partition is individually assessed for a positive or negative (digital...introduced how digital PCR works, let’s explain what kind of assays can be used to sniff out your desired ... through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous end joining...to generate translational reading frame-altering indels for “functional knockout” of a gene of interest...applications of precision genome editing, the remainder of this post will focus on assays to detect NHEJ... locus, as well as a reference probe designed to bind at an unedited site distal tothe predicted double...

  24. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...combination to find suitable antibodies for a target with very low expression, due to induced toxicity caused...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...time-consuming and expensive, it may be helpful to find an antibody that has already been validated for ...leukocytes. Where data is available from antibody-independent methods, like RNAseq or proteomics, it is possible...expression should have less antibodies labeling them. We find that cell tracker dyes are helpful in designing ....g. PMA or a specific cytokine) known to either induce or suppress the expression of a target of interest...

  25. Antibodies 101: Fab Fragments

    Type
    Blog Post
    Published
    Sept. 5, 2024, 1:15 p.m.
    ...’s antigen binding function but not the Fc region (more on when that might be later). Find scFv plasmids... region (or the “Fc”) and two Fragment Antigen Binding regions (or “Fabs”). The Fc region is the “tail...secondary antibody you should use when performing indirect immunoassays. The Fab regions, on the other hand...Fabs are the regions of the antibody that actually bind antigens.      Figure 1: IgG antibody structure...available different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced...hinge region disulfide bonds, resulting in two independent Fab fragments with small tails of the hinge region...above the hinge-region, which results in two independent Fab fragments. Size: ~50 kDa. Fv - These fragments...

  26. What's New in CRISPR - November 2021

    Type
    Blog Post
    Published
    Nov. 18, 2021, 2:15 p.m.
    ...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids Topics...gRNA and making strategic mutations in the DNA binding cavity of Cas12f. The CasMINI system is less than...active nuclease.  Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing with...to VP64. Read the paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing...AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   Selective endogenous encapsidation... The retroviral-like protein PEG10 specifically binds its own mRNA to facilitate its packaging into virus-like... other gene delivery. Read the paper in Science Find the SEND plasmids    Figure 1: SEND combines...

  27. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...antibody-specific antibodies bind to and recognize the constant region. Nonspecific binding, caused cell receptors... a different antigen; binding of each can be either simultaneous or independent of the other (Shahied,...that have the ability to specifically recognize and bind proteins. They’re used in a number of research and...final product. Because the constant (non-epitope binding) region is the first thing to go when downsizing...These reduced-in-size options often have reduced binding strength, half-lives, and/or specificity as well...of the variable region (the antibody region that binds to the antigen) from existing antibodies. Each cleaved...make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker, resulting...

  28. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    Published
    March 21, 2024, 1:15 p.m.
    ...through binding of the molecular species. Administration of these dyes results in direct binding between...fluorescent ligand when designing an experiment. You may find yourself defaulting to the most popular or familiar...experiment, including the accuracy and efficacy of findings. If you’re struggling to select the right fluorescent...with excitation/emission peak at 495/517 nm. FITC binds to an amine on a protein, enabling the labeling ...antibody, whereby excitation of blue light will indicate where FITC is bound through green light emission...dye-conjugation affects protein function and its propensity to bind cellular components. Some dyes target specific organelles...flexibility due to the affinity tag they typically bind to. Stability of fluorescence: For applications ...

  29. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    Published
    Aug. 9, 2022, 1:15 p.m.
    .../conditions can change binding ability – i.e., the antibody can no longer bind to the target). Unreliable...specific binding and has a strong signal. We call the unique pattern of the antibody binding “distinct...spent hours sifting through comments and papers to find the most reliable (and cost-effective) antibody ...causes of this unreliability: cross-reactivity (binds to non-target proteins), variability (separate batches...goldilocks antibody is highly specific (minimal binding to off-target molecules), reliable (wouldn’t perform... choosing which antibodies to screen, we kept in mind our own lab’s purposes. The Wu lab mostly uses whole-mount...Strength and accuracy The strength is based on binding specificity and exposure time. We first have a ...

  30. Hot Plasmids - October 2022

    Type
    Blog Post
    Published
    Oct. 6, 2022, 1:15 p.m.
    ... here. Here's what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated...Liu Z, et al. Cell. 2022.  Find JEDI-2p viral preps here (and here!)Find JEDI-2p plasmids here!  Liu,...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...EF1ɑ-driven viral vectors (AAV1) - and you can also find several other constructs in plasmid form. ...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...

  31. Fluorescent Proteins 101: Fluorescent Biosensors

    Type
    Blog Post
    Published
    May 18, 2017, 2:30 p.m.
    ...Visualization Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent...introduction to fluorescent biosensors, but keep in mind that there is a lot of variety in how biosensors...(CaM) and the chicken myosin kinase calmodulin binding region RS20. When calcium is absent, the construct...construct does not fluoresce; when present, calcium induces CaM/RS20 interaction, thereby changing the FP’s...FRET pair) separated by CaM and M13. When calcium binds, it triggers CaM/M13 interaction that increases ...same pattern described above - a sensing domain induces changes in a fluorescent signal based on ligand...biosensors to follow processes such as protein or DNA binding, enzyme or transcriptional activation, conformational...

  32. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    Published
    Aug. 3, 2023, 1:15 p.m.
    ... panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers are completely...a value of “1” indicating those markers are identical and completely indistinguishable. This tool is of...low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87; therefore... beads that will either bind any antibody (positive population) or won’t bind anything (negative population...panel, there are a few critical factors to keep in mind in order to get the best data possible.  Spectral...website has a convenient tool, called the similarity index, which allows the user to compare the emission spectra...designing a panel.     Figure 3: Similarity index across 12 different colors on a three laser spectral...

  33. Anatomy of a Plasmid Page at Addgene

    Type
    Blog Post
    Published
    Feb. 4, 2016, 3:30 p.m.
    ... Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...found yourself bamboozled by all of the different kinds of information on our plasmid pages? Well, to help... publication page, and finally on down to the individual plasmid page. The depositing lab page contains... in the collection, but you can use this name to find this plasmid from our search page (check out this...ways you can search our website). A quicker way to find this plasmid through search is... The Addgene plasmid...searching for, this is the quickest and easiest way to find it! But what are all those flames about? Flame...although you may have an entirely different use in mind! This section of our plasmid pages is relatively...

  34. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    Published
    May 18, 2018, 7:32 p.m.
    ...researchers do these things with nefarious goals in mind. Indeed, it’s not always obvious that we’re doing things... sciences and featured speakers from academia, industry, nonprofits, and publishing. The livestream video...preparation for my talk and at the minisymposium. You can find a variety of additional resources and information...that someone has replicated it precisely. You can find a great discussion of different types of replication...purposes we’ll call a study reproducible if its findings can be predictably applied to future work. Importantly...expect that every study should be reproducible. Indeed, researchers are pushing the limits of knowledge...reported by drug companies attempting to apply the findings from these studies, are quite low (<30%, Prinz...

  35. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    Published
    March 8, 2021, 2:41 p.m.
    ... membrane will bind to the proteins in the buffer, blocking the membrane  from binding to the antibodies...antibody binds to protein of the correct size. Many vendors will show that the antibody binds to transiently...slightly more popular due to its durability and high binding capacity. In addition, PVDF membranes can be repeatedly...unbound regions are “sticky” and have the potential to bind nonspecifically to the antibodies used for staining...But which antibody should you use? You will likely find several different antibody options targeting your...interest from a variety of vendors and you may even find plasmids encoding antibodies from Addgene that you...chains. The protein regions available for antibody binding, or epitopes, differ between a protein in its native...

  36. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...downstream of inducible promoters: the BxbI gene downstream of the anhydrotetracycline (ATc)-inducible promoter...sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies on a plate and... TP901 gene downstream of the arabinose (Ara)-inducible promoter (PBAD), and the A118 gene downstream ...downstream of the diacetylphloroglucinol (DAPG)-inducible promoter (PPhlF).  Output Plasmid: The output plasmid...register. Depending on the desired application, the inducible promoters we used to drive recombinase expression...register. RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output...promoters, and genes) were built with flanking primer binding sites (designed to have 20-30 nt length, ~60 oC...

  37. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    Published
    Feb. 18, 2016, 3:42 p.m.
    ...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating...generate compatible overhangs that allow them to bind. Ligase is used to make bonds between the insert...followed by the restriction sites EcoRI, XhoI, and HindIII. To place your gene in the proper orientation downstream...EcoRI site just 5’ of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you... backbone as well as the insert with EcoRI and HindIII and, when you mix the cut products together, the...two EcoRI digested ends will anneal and the two HindIII digested ends will anneal leaving the 5’ end of...

  38. Hot Plasmids: FRET-Based Biosensors

    Type
    Blog Post
    Published
    May 6, 2014, 1:07 p.m.
    ...high-affinity for calcium binding and developed a version of the protein with as few calcium-binding sites as possible...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the...and his colleagues began by isolating a calcium-binding protein, troponin C (TnC), from muscle cells. They...imaging for beginners Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans,...his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing...the transcription factor cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making...Metals Maarten Merkx of Technische Universiteit Eindhoven has similar interest in FRET-based sensor proteins...

  39. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ... crucial recognition region is disordered, Cas9 binds DNA very weakly, most likely through unstructured...regions.   Cas9’s transition from weak, random binding to precision gene editing requires three major ...3. Though Cas9 is not a static molecule in any binding state, RNA loading stabilizes the closed conformation... the open conformation (Cofsky et al., 2022), indicating that there is more to learn about this process...helix, purple; sgRNA, dark blue.   The REC lobe binds tightly to the repeat:anti-repeat duplex of the ... stem loops and the guide region. The stem loops bind along the outside of the protein, spanning both ...Figure 3 shows the guide RNA stabilizing the DNA-binding protein portion of the CTD, ready to check for ...

  40. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    Published
    July 31, 2020, 12:30 p.m.
    ...reversible and would avoid genomic off-targets or indels introduced through non-homologous end joining (...orthologs, and 7 Cas13c orthologs. They hoped to find a stably folded ortholog that cleaved RNA robustly...also increase off-target editing within the target window, possibly due to longer stretches of duplexed RNA...that editing is mediated by ADAR2DD, not PspCas13b. Find the plasmids for the RESCUE RNA editors here! Testing...duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of the tested constructs...system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note that RESCUE... RESCUE system small enough for viral delivery.  Find the RESCUE RNA editing plasmids here! Future directions...
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