...promoters, N-terminal tags, coding sequences of the gene of interest, C-terminal tags, plant terminators ...science lab.” Introducing GreenGate As Lohmann and his colleagues describe in the PLoS ONE paper that introduced... it no longer works, just trash it.” Lohmann and his colleagues including Joachim Forner now expect and...aptly named GreenGate – offers all of those same advantages to researchers working in plant model systems...

..., too! Bluegene is eager to decorate the lab but his short arms are causing him some difficulty… To give...celebrating the holidays and tweeting it with the tags #DeckTheLab and @Addgene. Christmas, Hanukkah, ugly...you name it, we want to see it.      All photos tagged with #DeckTheLab throughout the month of December...

...for any extrachromsomal hereditary determinant”. His proposal, however, was basically ignored. A separate...chapters on genome engineering, cloning, and plasmid tags, addtional content added to the "Plasmids that Glow...Learn more about the history of plasmids and cloning: CSHL Meeting: Plasmids: History & Biology The Joshua... history of plasmids...

...and gram negative bacteria periplasm. Efforts to tag endogenous genes for proteins in these compartments...and Lindsey M. Costantini! Erik Lee Snapp received his PhD in Molecular Microbiology and Immunology from...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz...the Department of Anatomy and Structural Biology. His interests include the quality control of secretory...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 ...FP-fusion proteins functionally relative to the untagged protein of interest. To avoid the accumulation...

... Replace the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long biotinylation... lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted...normally expressed in eukaryotic cells. Using this tag reduces the number of CAPTURE components that need...from three to two. Lentiviral delivery of BioTAP-tagged dCas9 and gRNAs. Using lentiviral delivery of the...system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate...conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). Think of 3C methods as taking...outperformed other 3D chromatin methods such as ChIA-PET or Hi-C, by increasing both the number of unique chromatin...

...contain a variety of N- and C-terminal tags (including His, S-tag, OneStrep, CBP, Trx, GST, Halo, MBP, ...electron microscopy (EM) tag providing superior subcellular localization of tagged proteins compared to traditional...distributing his Designer Receptors Exclusively Activated by Designer Drugs (DREADD) plasmids on his own, UNC-Chapel...promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and...of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated the use of the SpyLigase... risk of off-target effects. Harald Janovjak and his team at the Institute of Science and Technology Austria...expression levels. APEX2 is a monomeric, 27 kD genetic tag which can be fused to proteins of interest for EM...

...prokaryotes. Common Epitope Tags Tag Amino Acid Sequence FLAG DYKDDDDK HA YPYDVPDYA His HHHHHH Myc EQKLISEEDL...nucleotides. Epitope tags on the other hand are commonly used in molecular cloning to tag a gene within a ...E GAA, GAG Glycine Gly G GGU, GGC, GGA, GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Leucine...random incorporation of modified, fluorescently tagged bases onto the growing DNA strand in addition to...T, C, or G nucleotide. The 4 standard bases are tagged with a different fluorophore so they can be distinguished...when the polymerase incorporates a fluorescently tagged nucleotide. Because these special bases do not ..., the reaction is halted once the fluorescently tagged base is incorporated. Sanger sequencing requires...

...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... pMal plasmid (NEB) thus creating the pMal-T-Avi-His/BirA plasmid now available through Addgene. We have...Octet, Pall ForteBio). Tips for using pMal-T-Avi-His/BirA to add biotin to your protein of interest Grow...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...

...pAAV_hsyn_NES-his-CaMPARI2-WPRE-SV40 Syn CaMPARI2 his Constitutive 1 Schreiter 101061 pAAV_hsyn_NES-his-CaMPARI2...F391W-WPRE-SV40 Syn CaMPARI2-F391W his Constitutive 1 Schreiter 101064 pAAV_hsyn_NES-his-CaMPARI2-L398T-WPRE-SV40...L398T-WPRE-SV40 Syn CaMPARI2-L398T his Constitutive 1 Schreiter Calcium Sensor: jRGECO1 100852 pAAV.CAG.Flex.NES-jRGECO1a.WPRE.SV40...Sensors GRAB_NE nLight Oxytocin (OT) Sensors GRAB_OT Voltage Sensors Archon Voltron JEDI-2P ATP Sensors iATPSnFR2...-ChrimsonR-ST Syn jGCaMP8s none Cre-dependent 9 Histed 176749 AAV-Syn-H2B-jGCaMP8s-WPRE Syn jGCaMP8s none...GRAB_OTmut (control) none Constitutive 1, 9 Li Voltage Reporter: Archon 108422 pAAV-CAG-FLEX-Archon1-KGC-EGFP-ER2...KGC-GFP-ER2] Syn Archon1 EGFP Cre dependent 8 Boyden Voltage Reporter: Voltron 119036 pAAV-hsyn-flex-Voltron-ST...

...join one peptide tag to another peptide tag. Addgene: What is the advantage of peptide tags for building ...locking two peptide tags together. SpyLigase overcomes limitations in the use of peptide tags, which often form...Howarth: Peptide tags can be very generic; you should be able to use the same peptide tag for all sorts ...the National Academy of Sciences in March, he and his colleagues took another important step forward by...Natl Acad Sci U S A. 2014 Mar 17. PubMed) Peptide tag forming a rapid covalent bond to a protein, through...cancer in particular. They recently introduced the SpyTag/SpyCatcher system, based on a protein isolated ...Howarth: It came from our work in developing the SpyTag/SpyCatcher system, to be able to grab on to proteins...

...solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid... of a POI. As an investigator, you want a fusion tag to be inert, to not produce artifacts in your experiments...guest blogger Erik L. Snapp! Erik Lee Snapp received his PhD in Molecular Microbiology and Immunology from...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz...the Department of Anatomy and Structural Biology. His interests include the quality control of secretory...oligomers such as DsRed or high affinity FPs such as TagRFP. Problem 3 can be solved, in most cases, by using...

...illustrating the steps to fluorescently tag a C. elegans protein. The his-72 locus is shown as an example. Used...time. Creating a modular, selection-based gene tagging approach Multiple features of C. elegans have made... between a fluorescent protein (FP) and a 3xFLAG tag (see construct schematic below.) After self-excision...intron, and only the fluorescent protein and 3xFLAG-tag sequences are inserted into the genome. Figure...template plasmid to create fluorescent and 3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA...little hands-on labor and could facilitate the tagging of every protein in the C. elegans genome. Although... gene tagging...

...system for protein tagging. We spoke with Mendenhall and Myers about their new CRISPR tagging system, which...www.addgene.org/crispr/tagging. Addgene: What motivated you to adapt CRISPR for use in protein tagging? Myers: Our...epitope tag onto the transcription factors. By the way, you can actually use this for tagging any protein...Alabama in Huntsville explains it, a major goal in his laboratory is to understand the function of the non-coding...factors. Our groups got together to adapt CRISPR for tagging experiments and it’s already looking like it’s ...correctly, the second plasmid inserts a small epitope tag called FLAG at the end of the transcription factor...you can select the population of cells with the tagged transcription factor. The FLAG antibody has been...

... with containing chemical tags such as the SNAP-tag, Halo-tag, and CLIP-tag (Kohl et al., 2014) (Addgene...Takeshi Imai is a professor at Kyushu University. His lab has been studying functional and anatomical aspects... et al. "Ultrafast tissue staining with chemical tags." Proceedings of the National Academy of Sciences111.36... circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed PMID: 30454553....

...have two options:  Option #1: Affinity tag purification You tag YFP and use an affinity column for purification...protein. OptoNBs were also tagged with an infrared fluorescent protein tag which let the team detect its... used nickel-coated agarose beads coated with his-tagged OptoNBs. OptoNB coated beads were then imaged... be tagged, which limits these approaches to use with recombinant proteins. Purifying an untagged protein...Opto-Nanobodies (OptoNBs) purification You skip adding a tag to YFP and instead use OptoNBs. You fill a column...protein of interest in response to light allows for tagless protein purification as well as reversibly regulation...domain and a nanobody.  AsLOV2 is a light-oxygen-voltage sensing domain from the oat plant (Avena sativa...

...easy to harvest from cell culture media. Their His tags allow for further purification, if desired, but...RANbody is detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties...Spaghetti Monsters are highly antigenic tags that have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP... secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP, H2A2B Chicken IgY (Y) Labeled...lot-to-lot variability in activity. No Genetic Tagging - Since antibodies are produced in animals instead... via recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters (i.e. fluorescent...which are discussed in more detail below. The HA tag can be stained for with an anti-HA antibody and serves...

...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier...such as visualizing genomic loci using fluorophore tagged dCas9 may require the use of plasmid or viral-mediated...genomic deletions. What are the advantages of Cas9 RNPs? There are many advantages to using RNPs for CRISPR ... Delivering Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments using RNPs is... can be used to deliver a Cas9-gRNA RNP. This advantage has allowed scientists to perform in vitro and...transfection (B in the figure above), are in the early stages of development. David Liu’s Lab has demonstrated...

...from John Chodera's Lab - Get high expression of His-tagged human kinases in bactera. Listen to Our Segment...

...Allinen et al., 2004). In fact, Sanford H. Barsky and his team tested the influence myoepithelial cells had...touring musician needs? A backstage team. Meet the microenvironment, a tumor’s backstage team. Microenvironments... seem to take center stage in cancer development, the microenvironment backstage play just as important...breast tumorigenesis: Friend or foe?." Histology and histopathology (2018): 18021-18021. PubMed PMID: 29978449...microenvironment’s physiology is deeply connected to these 3 stages and contains multiple cell types: fibroblasts, ...

...concept include: Self-labeling tags (e.g., SNAP-tag, HaloTag, TMP-tag) – These widely used systems consist...strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion with your favorite protein...fluorophore-contianing “ligand” that binds to the tag. Like most good ideas in biological imaging, the ...genetically encoded tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure ...consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with a small... attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine...paclitaxel. Unlike other systems, no genetically encoded tag is required as the small-molecule binding motif targets...