...of biological contexts.  Proximity labelling relies on the labelling of neighboring proteins within a cell...methods.  BioID: a promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin... biotin ligase, BirA*, with a site mutation (R11G). This site mutation destabilizes the ligase, allowing...proximity labeling experiment! APEX: engineered ascorbate peroxidase that can rapidly biotin label proteins...promiscuous biotin ligase (Kim et al 2016). BioID2 consists of a humanized biotin ligase from A. aeolicus...increase the speed of proximity labeling by promiscuous biotin ligases (Branon et al., 2018). BioID and...BioID2 can take hours to label proteins and APEX2, although it can rapidly label proteins, requires H2O2...

... (Figure 1b). Engineered ligases (e.g., lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase...fluorescent proteins. In-cell labeling strategies The majority of in-cell labeling strategies have two parts... protein (Figure 1f). Labeling strategy pros and cons All of these labeling strategies have trade-offs...Overall, self-labeling tag systems (Figure 1b) are perhaps the best method for live cell labeling and the easiest... biochemists have developed techniques to make labeling chemistry easier and more functional in complex... imaging, the initial breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed...tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 1a). Other strategies ...

...restored, and nearby proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed... promiscuous version of BirA, an E. coli biotin ligase. In BioID, BirA* is fused to a bait protein and... than those which you’ve already fluorescently labeled. Figure 1: Using Split-BioID to Identify Protein...complementation assay where BirA*, an E. coli biotin ligase is split into two non-functional fragments: N-terminal... activity is restored, and nearby proteins are labeled with biotin. When proteins Y and Z part ways, BirA...Split-BioID, cells are lysed, protein extracted, and labeled protein interactors purified via streptavidin pulldown...including BioID, is its spatiotemporal controlled labeling of interacting proteins; nearby proteins are biotinylated...

...structures. Enzyme-catalyzed proximity labeling (PL) uses a promiscuous labeling enzyme and a small-molecule substrate...BioID was derived from biotin ligase and uses far less toxic biotin for labeling, but the process is much ... 2018 PubMed PMID: 29924347 Faster proximity labeling with TurboID and miniTurbo Article contributed...same directed evolution technique to the biotin ligase from which BioID was derived, creating a pair of... miniTurbo, which utilize non-toxic biotin, but label nearly as quickly as APEX2. What’s more, since the...

...RaPID) method. RaPID borrows the E. coli biotin ligase BirA* from BioID and allows a researcher to identify...that interact with an RNA of interest. Current labeling methods are not ideal for in vivo work. BirA* ...in vivo work. APEX2, a faster peroxidase-based labeling approach, is an alternative option but it’s toxic... methods rely on the promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within... optimum 18 hour time point, but one minute of labeling with BASU still generated a >30-fold enrichment...enrichment compared to E. coli’s BirA*. Faster labeling kinetics also means that BASU could potentially be used...Limitations of RaPID Like other BirA*-based proximity labeling methods, RaPID cannot distinguish between proteins...

...during PCR using a biotin labelled dUTP. There are multiple types of biotin labels that can be added to an...site of a newly synthesized enzyme. Biotin protein ligase (BPL) is responsible for this highly specific post-translational... for use in immunohistochemistry and antibody labelling. In these experiments, biotin is added to a primary...assay) Southern/Northern Blotting Cell surface labelling Attaching polypeptides to a substrate Flow cytometry...sequence is termed AviTag. Using AviTag, one can label a protein of interest with biotin by simply creating...both AviTag and BirA at Addgene! Oligonucleotide labeling Biotin can be added to either terminus of a DNA...depending on your downstream application. Biotin labelled oligonucleotides can be used in a multitude of...

... tag or labeling system. Image adapted from Lavis, 2016, under CC-BY. Common self-labeling tags Let...some popular self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from Roger Tsien...dynamic organelle interactions. a) Labeling scheme. b) COS-7 cell ER labeled with Halo-Sec61β with SiR-CA (...orthogonal self-labeling tags on different proteins of interest (or one self-labeling tag and a fluorescent...experiment (Figure 3). And using self-labeling tags makes updating your labels easy! Instead of taking the time...to talk about the power and flexibility of self-labeling protein tags. The basics These tags most often...fusion protein and add a specific ligand! A self-labeling protein gives you the flexibility to choose different...

...developed by Rui Tang in Monte Winslow’s lab to label cells after cell-cell interactions. Using G-baToN...sender cells expressing surface BFP generated BFP-labeled receiver cells. What’s more, G-baToN can be used...Skp1 and Cul3 proteins to form a SCF E3 ubiquitin ligase complex. In the presence of auxin, this TIR1 complex...chemogenetic system that brings Cas9 to ubiquitin ligase for degradation.      New from the viral service...

...for in vivo proximity labeling   By Ashley Waldron Proximity-dependent labeling has become a powerful ... Improving flexibility for in vivo proximity labeling Increased neuronal expression using Cre-mediated...pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et al., 2021)...Xiong et al. 2021.    BLITZ uses the biotin ligase TurboID fused to a conditionally stabilized GFP-nanobody...TurboID-nanobody protein and allows TurboID to start labeling proteins in close proximity to the GFP-tagged ...

...comes to labeling cells for flow cytometric analysis, the most common method is a cell surface label, where...alongside standard surface labels. Here, I will introduce you to a few useful labeling and staining techniques...techniques beyond standard surface labeling. Pro tip! Many people use 'labeling' and 'staining' interchangeably...intracellular labeling must be performed only after viability staining and surface labeling are already ...nuclear fix/perm buffer to label intranuclear proteins of interest, you can label cytosolic proteins alongside...intracellular labeling cells need to be fixed and permeabilized prior to incubation with the labeling antibodies...covered in this post. Besides direct labeling, indirect labeling can be used when suitable fluorophore-conjugated...

...biology studies. For example the E. coli biotin ligase, BirA, has been used to identify and study protein-protein...protein-protein interactions in proximity labeling techniques such as BioID (Roux et al., 2012) and APEX...

... different fluorescent labels. The machine detects which fluorescently labeled nucleotide is present at...combined discoveries of restriction enzymes, DNA ligase, and gel electrophoresis allowed for the ability...strands of varying lengths whose last nucleotide is labeled. Once all the newly synthesized DNA is made, the... of DNA is first fragmented into small pieces, labeled with a short DNA barcode, and amplified. These ...base is added. Again, each base (A,C,T, or G) is labelled with a different color making it easy to identify...and the machine cannot distinguish between the 4 labelled nucleotides. Epitope tags on the other hand are...

...B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha competent...concentration, use 1 μL) 2 μL 10x NEB T4 DNA ligase buffer 1 μL NEB T4 DNA ligase add ddH 2 O to bring to 20 μL final...should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate puromycin-containing...

...visualize chromatin and RNA dynamics in live cells. Labeling can be done by fusing catalytically inactive dCas9...protein (GFP), creating a customizable DNA or RNA label for fluorescence microscopy. RNA aptamers, like ...genomic locus requires recruitment of many copies of labeled proteins to the site. Chromosome-specific repetitive...proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus...RNA in live cells, including orthogonal RNA/DNA labeling in the same cell. Figure 11: Overview of visualizing...acceptor site to dCas9 and co-expressing BirA biotin ligase. Whatever the chosen epitope tag, the locus is ..., S., & Pederson, T. (2015). Multicolor CRISPR labeling of chromosomal loci in human cells. PNAS . 112...

...visualize chromatin and RNA dynamics in live cells. Labeling can be done by fusing catalytically inactive dCas9...protein (GFP), creating a customizable DNA or RNA label for fluorescence microscopy. RNA aptamers, like ...genomic locus requires recruitment of many copies of labeled proteins to the site. Chromosome-specific repetitive...proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus...RNA in live cells, including orthogonal RNA/DNA labeling in the same cell. Figure 11: Overview of visualizing...acceptor site to dCas9 and co-expressing BirA biotin ligase. Whatever the chosen epitope tag, the locus is ..., S., & Pederson, T. (2015). Multicolor CRISPR labeling of chromosomal loci in human cells. PNAS . 112...

...deposited a smaller version of the promiscuous biotin ligase used in their popular biotinylation method for ...This method uses a bait protein fused to a biotin ligase to biotinylate proximate proteins in the cell. ...proteins, requires less biotin, and shows enhanced labeling of proximate proteins. Overall BioID2 improves...