...lentiviral vectors to stably or inducibly express tagged iPAK4 from different promoters, specific time points...plasmids here! Lin D., Li X., et al. (2023). Time-tagged ticker tapes for intracellular recordings. Nature...off-target effects of gene therapy, Feng Zhang’s lab has developed a system called DIRECTED (Delivery to Intended...on-target delivery with various delivery modalities and has the potential to reduce negative side effects. This... assays like IHC. In fact, Anti-PSD-95 [K28/43R] has been one of the hottest antibodies in Addgene’s collection... collection since we began distribution and has been verified to perform well in standard IHC and western...externally-timed bands of color with HaloTag-iPAK4 by applying different HaloTag dyes and showed they could record...

... fusion tag to be inert, to not produce artifacts in your experiments. Considerable effort has gone into...solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid...world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize...dimerizing protein of interest fused to a dimerizing FP has potential to form large stable polymers of dimers... monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are designated ...oligomers such as DsRed or high affinity FPs such as TagRFP. Problem 3 can be solved, in most cases, by using...

...this technology and its advantages? Fujii: In the last several years, my lab has been working on development...immunoprecipitation (ChIP) (iChIP) technology, which uses locus-tagging and affinity purification, in 2009. However, iChIP...generated a series of retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system...joined Addgene's Advisory Board.  Addgene: Your lab has worked extensively with enChIP systems. Can you describe... systems to Addgene in the near future. Addgene: Has your lab been working on any other technologies that...plasmids. The merit of depositing plasmids to Addgene has been spectacular. We've got more than one hundred...

...fluorescent proteins (GFP and YFP) and protein tags (c-MYC and HA). We have also created the Monoclonal Antibody...community. Thanks to Our Guest Blogger!   David Soll has been the Director of the Developmental Studies Hybridoma...

... Pages. Addgene sequences the plasmid to verify tags, mutations and a portion of the insert, but we do...provided by your sequencing provider. My sequence has “N”s in it- what does that mean? “N” is the letter...

...cut, activate, interfere, visualize, nick, purify, tag, scaffold, CRISPR-display, and dCas9-FokI. This table...Alternatively, you can select a target sequence which has already been shown to work in CRISPR experiments....experiments. Addgene’s dedication to resource sharing has led us to develop a searchable and sortable datatable ... to the fact that every gRNA listed in the table has been used effectively in a published article. This...target sequence, and model system. Even if a gRNA has been demonstrated to work in your system, it is worth...that locus. The data in the validated gRNA table has been derived from scientists’ submissions, either...

...generate one molecule of GFP-tagged LC3 for every one molecule of RFP-tagged LC3∆G. Like the unmodified ...number of autophagosomes. This approach, however, has inherent limitations because it turns a dynamic process...

...Fluorescent Protein Should I Use? However, fusion proteins tag your protein of interest with a fluorescent reporter...protein. One downside of fusion proteins is that the tag can alter the structure of the protein. In some cases...timing of expression can also be affected by the tag.  Protein expression, measured using antibody conjugated...measured using visible light scatter The optical system has both visible and fluorescent light sources. Visible...autofluorescence from your target cell type or that has arisen from fixation. Also include an isotype control...secondary antibody control in which the primary antibody has been omitted from the staining protocol. This control...gating during data analysis.  We hope that this blog has provided you with an overview of flow cytometry for...

...BirA* biotinylation activity ceases. After biotin tagging of proteins in both BioID or Split-BioID, cells...depending on which terminus of the bait proteins were tagged but also which fragment of BirA*, either the N-...N- or the C-terminus, the bait proteins were tagged with. It’s best to test all combinations and pick ...opposed to an individual protein. The Bethune Lab has overcome this limitation by creating Split-BioID,... of the fusion protein. Advantages of Split-BioID Split-BioID’s key advantage over other PPI methods, ...proximity-dependent biotinylation technique BioID. The key advantage of Split-BioID is that it allows for the validation...

...terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or affinity tags (HA, FLAG)....putative T1 transformants: When your T1 is on soil and has recovered from the stress of selection you can clip...cell fate maintenance in the stomatal lineage and has made countless transgenic plants in the process. ...

...rate. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would be... Even though GFP has been so thoroughly characterized, it turns out this protein has a few more secrets...photoconvert GFP from green to RED! This simple technique has been shown to work in plant, Drosophila and mammalian...is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants used by Sattarzadeh...

... Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs... can be used in cells or organisms in which Cas9 has been integrated into the genome. gRNA Empty Vectors...Marraffini pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) 47868 Mammalian U6 yes, cut N. meningitidis...pyogenes tagRFP Ebert pL-CRISPR.SFFV.tRFP 57826 Mammalian/Lentiviral BsmBI yes, cut S. pyogenes tagRFP Ebert...Mammalian/Lentiviral BbsI none S. pyogenes Puro, TagBFP Yusa lentiGuide-Puro 52963 Mammalian/Lentiviral...Mammalian/Lentiviral BsmBI yes, cut S. pyogenes tagRFP Ebert pFGC-pcoCas9 52256 Plant AscI, PacI, SbfI...57824 Mammalian/Lentiviral BsmBI none S. pyogenes tagRFP657 Ebert pL-CRISPR.SFFV.GFP 57827 Mammalian/Lentiviral...

...fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated into Nanoblades, just like...insertions like the addition of a fluorophore or tag. To utilize HDR, a donor template containing the ...particles with polybrene, a cationic polymer. Polybrene has long been used to increase retroviral transduction...of mouse zygotes and mice with Nanoblades CRISPR has also been extensively used to create transgenic mice...

...measurable through an epitope tag such as FLAG/His/GFP. Fluorescent protein tags, which correlate closely ...different epitopes. Detection of overexpressed (tagged or untagged) protein When expression plasmids are available...expensive, it may be helpful to find an antibody that has already been validated for flow cytometry through...Characterization through Open Science) initiative has now started producing flow cytometry characterization...overexpression. Additionally, if the cell line used has endogenous levels of expression, this can make the...TIM-1 (Single pass type-1 membrane protein), which has been designated CD365 in the most recent HLDA workshop...as part of the YCharOS open science ecosystem. It has received in-kind support to produce this data from...

...fluorophore-tagged anti-rat IgG1 secondary to detect Protein A and green fluorophore-tagged anti-mouse ...imaging experiment   We hope that this blog post has answered all of your questions about the utility ...

...controlled easier than formation of GST dimers. Avi-Tag plasmid for improved biotinylation selectivity A ...fusion protein from a bacterial extract. The MBP tag can be removed using Xa protease. The carboxyl-terminus... binding protein (MBP, New England BioLabs). MBP has a similar function as GST in that it provides a method...amino acid) AviTag sequence in a protein and adds biotin to a lysine residue within the AviTag. However,...carboxyl-terminus of the fusion protein includes an AviTag and a 6X HisTag for affinity purification (see plasmid ...dramatically increased accessibility of the AviTag and HisTag, which were “buried” in a few of our proteins...fusion proteins - but it would remove both the AviTag and HisTag. We were surprised that we couldn’t find ...

...after the first tagging step. As an additional benefit of plexWell™’s sequential tagging procedure, the...with our plasmid assembly pipeline. To date, this has resulted in high quality NGS-based assemblies for...

...is the ability to follow multiple fluorescently tagged species simultaneously. To do so with fluorescent...generally the most detectable and so should be used to tag your least abundant proteins, with the blue and infrared...protein, if you know that your protein of interest has a similar spectrum. For example, here's a screenshot...#49004) to the spectra of both mCherry and TagRFP.  Here, the TagRFP spectrum is shown in the darker colors...peak of the TagRFP excitation and the emission filter collects a larger fraction of the TagRFP emission ...with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an infrared... emission. For this filter set, we would expect TagRFP to give a brighter signal than mCherry. In general...

...CRISPR plasmids at Addgene! Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand...incorporating very long ncRNAs is impractical. Instead, tags or stop elements can be inserted to disrupt the ...CRISPR systems for targeting ncRNAs While CRISPR/Cas9 has proven to be an extremely useful system for modifying...This system is often used to knock down lncRNAs and has fewer off-target effects. Newer Cas protein variants...transcriptional activators such as VP64, SAM, or SunTag to specific ncRNA loci to enhance or upregulate...

...short sequences of up to 100 bp, such as epitope tags, design primers with template binding regions to...combining multiple primers, a C-terminal epitope tag can be moved to the N-terminus of a protein-coding... can be introduced in a gene, while adding a GFP-tag (Figure 3). Achieving multiple modifications requires..., TOP10, and Mach1. No common lab cloning strain has failed so far. What is most important is the transformation... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...modification, from insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer...rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase...