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We narrowed to 527 results for: HA tag

Showing: 151 - 180 of 527 results

  1. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

    Type
    Blog Post
    ...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...

  2. Tips for arabidopsis transformation

    Type
    Blog Post
    ...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. ...

  3. Seeing Red: Simple GFP Photoconversion

    Type
    Blog Post
    ...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...

  4. A Look at Addgene's QC Process

    Type
    Blog Post
    ...plasmid they ordered contains the correct inserts, tags, promoters, and other critical functional components...on the information provided and vector type  Any tags, selectable markers, and fusion proteins listed ...including the propagation of plasmids in bacteria — has many points at which small errors can be introduced...Catching and addressing QC issues Addgene’s QC team has developed a process for identifying, categorizing...

  5. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    ...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...

  6. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...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...

  7. The Power Behind NGS Plasmid Validation: seqWell

    Type
    Blog Post
    ...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...

  8. In Vivo Biotinylation of Bacterial Fusion Proteins

    Type
    Blog Post
    ...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 ...

  9. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ...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 ...

  10. Choosing Your Fluorescent Proteins for Multi-Color Imaging

    Type
    Blog Post
    ...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...

  11. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...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...

  12. CRISPR Plasmids - Empty gRNA Vectors

    Type
    Collection
    ... 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...

  13. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ...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 ...

  14. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...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...

  15. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...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...

  16. Mapping the 4D nucleome with CRISPR/Cas9

    Type
    Blog Post
    ... 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...

  18. New and Upcoming Viral Vectors - Spring 2019

    Type
    Blog Post
    ...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...

  19. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...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...

  20. Lentivirus Plasmids

    Type
    Collection
    ...randomly into the host genome, it has the potential for insertional mutagensis. The 2nd and 3rd generation ...

  21. Optogenetics Guide

    Type
    Guide
    ...function. TULIPs Tunable light-inducible dimerization tags (TULIPs), plasmids allow cloning of protein coding...sequences with GFP-LOVpep, cpPDZ, ePDZb and ePDZb1 as tags. pDawn/pDusk Blue-light photoreceptor for light-...turning on and off the light source. Optogenetics has been broadly applied to study the physiology of the...channel. ChR2 is preferred over ChR1 because ChR2 has higher conductance at physiological pH and trafficks...Depolarizing) ChR Variants. The optogenetic toolbox has been expanded by scientists through both the identification...heterodimerization can reconstitute the protein. This principle has also permitted the design of synthetic two-part transcription...Phytochrome, cryptochrome, and LOV (light oxygen voltage)-based systems have been used in many experimental...

  22. Luminescent Imaging with Nano-lanterns

    Type
    Blog Post
    ...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...

  23. Fluorescent Protein Travel Awards - FLiPs and Fluorescent Protein Biosensors

    Type
    Blog Post
    ...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 ...

  24. New and Upcoming Viral Vectors - June 2019

    Type
    Blog Post
    ...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...

  25. Another Pathway into Cells: iTOP

    Type
    Blog Post
    ...surprising discovery that both purified CPP-tagged protein and an untagged control could enter cultured cells ...derived from the same cell type; however, their usage has been limited by technical problems. For instance,...binary systems in which transient protein expression has a long-lasting effect, such as Cre- or Cas9-mediated...effects. Third, protein purification (once a protocol has been established) may be less laborious than viral...used for protein purification must be helping the untagged protein enter cells. Subsequent work found that...stem cells, they found very high recombination percentages (78-79% after two rounds of transduction.) Compared...

  26. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...cells according to whether or not they have been tagged with a fluorescent protein or dye. It separates... distinguishing cell populations that have been tagged with different fluorescent proteins or dyes. 3.... cell mobility within these complex environments has never been easy and, for many years, researchers ...Intravital imaging powered by two-photon microscopy has uncovered host-pathogen interactions leading to the...proteins can be used in your experiments, experience has shown that some are more reliable and more efficient...important to finely tune the excitation wavelength as it has been shown that even a small incremental change of...this system called Brainbow, the Ubow mouse strain has been created to fate map Langerhans cells and follicular...

  27. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ...into a vector with a large tag at the N-terminus, but you don’t have the tag already included in the vector...you can insert both the gene of interest and the tag sequences into the vector in one step without scars...where the vector that you want to use is very large, has a high GC content, contains a lot of repeats--any...In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using... common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning...thereof) nor a serious time commitment. Another advantage is that this process makes it easy to generate...will be using. Some of these products may offer advantages that Gibson assembly does not such as increased...

  28. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ... be selected, since it cleaves at the stop codon TAG. Design your repair template with 55-100 base...mScarlet).   Plasmid ID Plasmid Name Tags 69090 pMJ915 MBP (N terminal); 6xHis (N ...CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including...including creating Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method...left homology arm guanine (i.e. GG); this design has been shown to increase T7 promoter activity. To avoid...

  29. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi

    Type
    Blog Post
    ...respectively. mNeonGreen can be used as a fusion tag for a wide range of applications from traditional...single-molecule super resolution imaging. mNeonGreen also has little sequence identity to other common fluorescent...fold brighter than EGFP and is photostable. It also has narrow excitation and emission peaks (max excitation...was then screened through successive rounds of mutagenesis for the brightest teal fluorescent proteins that...scientists from Florida State University, they used a mutagenesis approach and found that specific amino acid changes...

  30. Hot Plasmids December 2018 - C. elegans RNAi, TurboID, Detecting Viral Outbreaks, and Protein Binding Specificity Tools

    Type
    Blog Post
    ...used for PL were APEX2 and BioID. APEX2 is fast, tagging proximal proteins within minutes, but requires ...being targeted via RNAi. Recently the Vellai lab has created an improved RNAi vector that leads to significantly...sequence of the toehold switch. For example, Green has also developed toehold switches to detect Zika virus...protein motifs than previously thought. Sidhu’s group has made several hundred SH3 domain containing plasmids.... In addition to these constructs, the Sidhu lab has made hundreds of additional plasmids available through...infect millions annually. Because it is highly contagious, it is essential to contain outbreaks when they...
Showing: 151 - 180 of 527 results