...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...

...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 ...

...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...

...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 ...

...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...

...The antibodies are conjugated with a reporter or tag that can then be detected.  Typically, the reporter... (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we will discuss...testing a recombinant protein, especially one with tags, be sure to include a sample that expresses the ...While the method is simple and saves time, it also has several drawbacks. Direct ELISAs generally have higher... of the advantages and disadvantages of each ELISA method Method Advantages Disadvantages Direct...rundown of the different types of ELISA, advantages and disadvantages of each, and some considerations when...isotype.     Specificity and timing are the major disadvantages of the indirect method. The assay only uses ...

...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...

...and gram negative bacteria periplasm. Efforts to tag endogenous genes for proteins in these compartments...Munro, Sean, and H. R. Pelham. "Use of peptide tagging to detect proteins expressed from cloned genes:...Avaliable at Addgene Read Our Blog Post on Protein Tags Read Other Posts on Fluorescent Proteins Browse ...enabled numerous cell biology assays. However, there has been a pervasive view that most FPs in a cell will...FP-fusion proteins functionally relative to the untagged protein of interest. To avoid the accumulation...

... from this publication at Addgene. A protein-tagging system for signal amplification in gene expression...nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional...transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently label a sequence...imaging time for this technique. The NIH Common Fund has made mapping the 4D nucleome a specific priority,...ability to label live cells make this system truly advantageous. This new technique, developed in Thoru Pederson...sensitivity of the technique. One potential solution is SunTag, a synthetic scaffold that can be used to recruit...from Addgene!) Since the signal level provided by SunTag is so high, cells may be imaged under lower illumination...

...of them containing fluorescent markers or protein tags, and suitable for molecular cloning. Plus, we decided...transformation?  Great news. A subset of our collection has been prepared as part of our new cloning grade DNA...

...AAV5  pOTTC1596 - pAAV SYN1 HA-hM3D(Gq) 121538  AAV5  pOTTC1484 - pAAV SYN1 HA-hM4D(Gi) 119741  AAV5 ... vectors for experiments encoding a fluorescent-tagged molecular tool.   Find pAAV-FLEX-tdTomato, pAAV-GFAP104...iGABASnFR GABA sensor (pAAV.hSyn-FLEX.iGABASnFR) has good membrane localization and brightness. The F102G...its F102G variant in AAV1 at Addgene! Archon1, a voltage sensor AAV Historically, electrical activity in...measured with implanted electrodes. The Archon1 voltage reporter allows a way to monitor neuronal electrical...Archon1-KGC-GFP-ER2]) of this membrane-embedding voltage detector. Find Archon1 and its cre-dependent version...pAAV-hSyn-EGFP *Also coming in our new 20 ul trial size! Voltage indicators Plasmid Serotype Name 119036  AAV1...

...pair changes, and the addition of protein tags. It also has the flexibility to modify the E. coli chromosome...non-selectable DNA fragments, such as gene replacements and tags.  The typical frequency of recombinants is 1 positive...recombineering system The lambda red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and... in the center of the sequence. Since lambda red has a higher recombination frequency when the lagging...activating MMR: 1) use a strain of bacteria that has key MMR proteins knocked out or 2) specially design...recombinant clones If a antibiotic resistance gene has been inserted, recombinants can first be selected...repress expression of exo, beta and gam.  A major advantage of using this method for lambda red expression...

...Plasmids Find Empty Backbones with Fluorescent Protein Tags  ... first publication in 2012, the Nagai laboratory has assembled a collection of multicolored nano-lanterns... low luminescence By itself, Renilla luciferase has a low quantum yield, and when transiently expressed...using chemiluminescent proteins for imaging, but it has precluded widespread adoption of the technology. ...Nano-lanterns and your experiments The Nagai laboratory has a done a wonderful job characterizing Nano-lanterns...described the first Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent...

...AAV2 pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine 121538  AAV5 pOTTC1484 - pAAV SYN1 HA-hM4D(Gi) 121539 ...for activating and inhibiting neurons. Minimally-tagged DREADD AAV from the Richie lab are especially useful...121539  AAV5 pOTTC1596 - pAAV SYN1 HA-hM3D(Gq) New chemogenetics AAV Plasmid Serotype Name 119741  ... AAV9 pAAV-hSynapsin1-FLEx-axon-GCaMP6s New voltage sensors Plasmid Serotype Name 119036  AAV1...

...fusion protein consisting of sfGFP fused to a His-tagged ABC transporter known to bind to the HMO of interest... dronpa fusions. With these various designs, Xin has been able to develop photoswitchable Intersectin,...Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5, and Cas9. Xin has demonstrated the utility of these FLiPs in a variety... of cells that actively produce the HMOs. Fatima has demonstrated the successful production of both types...precisely spatio-temporally controlled as a key advantage over other inducible enzyme technologies. She ...