...right negative control is critical to the success of BiFC experiments. The ideal control is a mutated variant...-to-transfect cell lines where plasmid dosage can be tightly controlled. This is also theoretically possible...because it relies on a single BiFC pair. Find BiFC plasmids for your experiment at Addgene! How to Design ... work. You will also need to generate negative control constructs — more on this later. For more on constructing...check out our article: Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular... measure the fluorescence of a single FP. Your control construct may generate a modest level of fluorescence...intensity of your BiFC constructs to your negative control to determine whether your PIPs actually do interact...

...blot showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common... be used for. Addgene’s Antibody Plasmid Collection includes plasmids that express antibodies, nanobodies...immunoglobulin genes, recombinant antibodies are plasmid-based. Their sequences can be optimized for specificity...protein (Wang et al., 2013). These proteins are also plasmid-derived. However, a disadvantage to ScFv is their...nanobodies, or synbodies, can be produced from plasmids, saving time and money. Nanobodies and synbodies...detection. You’ll also want to use the appropriate controls for your experiment to account for potential non-specific...quantitatively if you also load the gel with appropriate controls for normalizing the protein concentration. To ...

... from the default GFP (green) to RFP, CFP, or YFP fluorescence. In Brainbow 2.1, GFP is tagged with a ...and activate fluorescence of the downstream OFP, GFP, or RFP. Cells that express the construct but do ...use in Drosophila melanogaster and is under the control of Flp recombinase. The XFPs in Flybow are membrane-tethered...a specific tissue. XFPs are introduced using a plasmid construct called Zebrabow-GateDest containing an...generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic RFP...introduced in vivo either by electroporation of plasmids, or using AAV vectors. The Imai lab also developed...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically low...

...Using an epifluorescence microscope and GFP fusion proteins controlled by promoters specific to the immune... to track where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells ...enabled by fluorescent proteins The optimization of GFP and its derivatives by the late Roger Tsien changed...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes... the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after infection...researcher can flank fluorescent proteins in a plasmid with loxP sites such that their expression is turned...Check out Our Fluorescent Protein Guide Pages Find Plasmids for Subcellular Localization Find Fluorescent ...

...hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~...infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction...types they tested. Interested in using Nanoblades? Plasmids are available from Addgene. Genome editing primary...to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line with previous research...editing in their livers, while non-injected and control gRNA injected mice had no detectable editing. Additionally...out our comprehensive CRISPR guide Find CRISPR plasmids and resources Learn about viral production ...

...fluorescent protein reporter under the control of promoters that control cell cycle gene expression.  Additional...-up approach will allow me to create a fully controllable and reproducible set up,” Bernach explains. ...containing unstable fluorescent proteins under the control of promoters responsive to water or nutrient levels...fluorescent protein topics page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org...Addgene.org Find fluorescent protein biosensors Browse Plasmids from the Tsien lab Browse the Michael Davidson...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...a variant has wild-type levels of abundance, the eGFP will be stable and fluoresce. But if the variant...

...from the Church lab uses this system to control expression of GFP.)  To turn this inducible system into ...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...promoters can be regulated via positive or negative control. Positive Repressible: An activator protein is ...system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA). They fused TetR with the...Tet-Off system.  For more information on Addgene Tet plasmids, see our Tetracycline resource page. Cumate Switch...interest to be expressed. These permit exquisite control of gene expression and tracing of gene expression...that sometimes it is necessary or desirable to control or look at multiple genes at the same time.  The...

...stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation ...GreenGate Plasmids Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More Plasmids 101: Origin...Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into...in two species: Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned...It’s a lot of information to fit into a single plasmid, so most first-generation binary vectors are quite...Arabidopsis, and corn. For both β-glucuronidase (GUS) and GFP reporters, as well as multiple transient and transgenic.... The kanamycin antibiotic resistance gene is controlled by a weaker constitutive Nos promoter, and it...

...Reporter Gene like Luciferase and GFP Resources on Addgene.org Find Plasmids for Your Research     ...Once you've gotten to know what a plasmid is in general, it's time to take a look at some of its parts...origin of replication are necessary to replicate plasmids and make sure the cells maintains it. Here, we'll...the promoter, which is necessary for getting the plasmid express our gene of interest. This element is responsible...target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription.... This means that the promoter present in your plasmid backbone must to be compatible with the type of...transcript, you will also need to make sure your plasmid has a promoter suited to working in your host organism...

...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes...crossover event between the vector plasmid and the packaging plasmid caused the Psi sequence to be placed...expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone your gene of...usually 293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3) and one with either tTA or rtTA (FUW-M2rtTA...techniques are still used and efficiently transfer plasmid DNA efficiently into dividing immortalized cells... (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding the insert of interest as well as sequences...

...cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid... One such control is dividing up the essential components of a virus into different plasmids. For a retroviral...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection...virus and its infectivity in check. Engineering controls, safety systems that are built into how something... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety...

...of the new plasmids, antibodies, and viral preps in the repository through our hot plasmids articles. ... blot for the myc-tagged protein GFP-smFP-myc expressed from Plasmid 98926. See the Applications section...Think we missed something hot? You can pitch a Hot Plasmids section (antibodies and viral preps welcome!) ... you can also find several other constructs in plasmid form. Fig. 1: Summary of the approach and...JEDI-2p viral preps here (and here!)Find JEDI-2p plasmids here!  Liu, Zhuohe et al. Sustained deep-tissue...represent a long-sought tool for researchers to control K+ gradients and can be used as a potent optogenetic... improve the performance of HcKCRs. Find HcKCR plasmids here!   Govorunova EG, et al. Kalium channelrhodopsins...

...spatiotemporal control over labeling methods as compared to transgenic animals (17).  These viral plasmids are available...mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories later...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed...University of Miami Miller School of Medicine. Find plasmids from the Lee Lab here. Pantelis Tsoulfas is an...University of Miami Miller School of Medicine. Find plasmids from the Tsoulfas lab here.     References: H... nerve afferents labelled with a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical...

...their Cas9 and gRNA backbone plasmids with Addgene. Additional control plasmids for the TKO library will ...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...

...protein. With this control set, you should see your target protein IP’d in the positive control sample but not...permanently altering the structure of the protein.  Controls As with any experiment, it is critical to include...include proper positive and negative controls for an IP. When possible, perform the IP in parallel with ...interest and a sample that does not. Common negative controls include knockout cell lines or tissues that do... not in the negative control, confirming that the capture antibody is functioning as expected. In order...perform parallel IPs with beads only and an isotype control antibody. Alternative applications Oftentimes, ...In cases such as this, an epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest ...

...placing FP timer DsRed-E5 under the control of Neurog3 (a gene controlling pancreatic differentiation) in ....  Table 1: Monomeric FP Timers and Associated Plasmids from Subach et al. Protein Excitation (nm) ...) Emission (nm) Brightness pKa Maturation Plasmids Fast-FT 403 (blue), 583 (red) 466 (blue), 606 (red...synthesized or long-lived transcription factors control gross changes in gene expression as new body sections...Pratik, et al. "A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive...Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get Advice on Choosing Your Fluorescent Protein...

...transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants into a ...in this case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts with a transcriptional...reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu Zhang’s lab) is an example...accounted for using appropriate replicates and controls (see Tips below), and by validating leads with...reporter gene activation. Including a prey-only control also provides a baseline level of reporter activation...that are known to bind serves as a good positive control to ensure that your setup works. The last thing...Yeast Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About Yeast Vectors Read our...

....org Find plant plasmids Visit our plant resource page Deposit arabidopsis plasmids with Addgene ...this method include: Cloning and transforming a plasmid into the bacterium Agrobacterium tumeficans - a...available (Weigel and Glazebrook, 2002). Find plant plasmid resources at Addgene  Choosing an expression system...options, but here are a couple of my favorites. Plasmids from the Tsuyoshi Nakagawa lab are binary gateway...terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or affinity tags (HA, FLAG). There...prefer Golden Gate assembly, there are a series of plasmids from the Dinneny lab that additionally allow for...culture you are growing definitely contains your plasmid. Grow lots of Agrobacterium: The more agrobacterium...

...protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all viral genes...going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC and... has been inundated with requests to share our plasmids with other researchers. We have been excited to...Gordon) have been quite busy shipping and sending plasmids to all who request them -- in locations all over...Facebook. Find more COVID-19 and coronavirus related plasmids and resources at Addgene* Many thanks to our...people, not just scientists! *If you are requesting plasmids for coronavirus or COVID-19 research, please email...

...You’ve designed the perfect experiment – controls, conditions, and everything in between – now all you... (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...Antibody protocols  Resources on the Addgene blog Plasmids 101: Protein tags Antibodies 101: Epitope tags...