...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...

...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...

...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...

...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...

...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...

...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...

... 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 ...

...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’...

...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 ...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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,...

... 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...

...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...