...interactions scientists have long used antibody based affinity purification and mass spectrometry approaches...scientists have developed newer methods termed proximity-based labeling to better capture these interactions in..., which can be a challenge for classic antibody based affinity purification/mass spectrometry methods....efficiently label neighboring proteins The first biotin based proximity labelling technique, BioID, was developed...these issues, the Ting lab utilized yeast display-based directed evolution to generate two mutants, the ...interaction detection) uses proximity protein labeling based on BioID to identify RNA-protein interactions (Ramanathan...Trinkle-Mulcahy, L. (2019). Recent advances in proximity-based labeling methods for interactome mapping. F1000Research...

... AraC transcriptional regulator Their system is based on the AraC protein that controls transcription ...pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid. It does not include the ...the PBAD promoter. If you already have a pBAD33-based plasmid expressing your gene of interest, all you ... created by the team (Fig. 2). These images are based on the concept of bacteriographs created about a...

...which was technically challenging. Other homology based technologies We've described Sequence and Ligation...to SLIC and Gibson, there are yet more homology based assembly methods that have been described--CPEC ...store-bought cloning kits available which are all based on long-overlapping regions, have no requirements... these days. Gibson and the other long-homology based cloning methods are useful alternatives to the standard...assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009; 6, 343-345. PubMed PMID: 19363495...

...off-target edits of base editors, which convert a nucleotide base pair to another base pair at a specific...Bhoobalan-Chitty Y, Peng X (2018) Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus...CRISPR activity is an important step towards CRISPR-based therapies and for more precise editing in the lab...

...DNA sequence. Base Editors: Modified versions of CRISPR nucleases that make single base changes in the...includes the details on advances in CRISPR including: Base editing Prime editing Anti-CRISPRs … you name it...

... proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence...target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat section of the crRNA, forming...interchangeably with the term sgRNA. Seed sequence: The 8-10 bases on the 3’ end of an RNA guide (either crRNA or sgRNA...Sheet  Resources on the Addgene blog A Needle in a Base-Stack: Cas9 Structural Biology...

...the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal ... expression in yeast. These tagging vectors are based on previously described pFA6a-link vectors and include...stability, and disruption of the tagged protein. Based on their findings, the authors recommend optimal...the artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University of...

... in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium is king in...Neurotransmitters Many canonical fluorescent as well as FRET-based biosensors have been engineered to detect a wide...ATP ratio. Most of these sensors are fluorescence-based and have been optimized for a wide-range of uses... pair on actinin will produce different signals based on changes to the structure of actinin in low vs...Biosensors Illuminating Epigenetics with a FRET Based Biosensor Fluorescent Proteins 101: Fluorescent...

...the first thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal...this too. It's just that for non-neural cells, ion-based communication happens at a slightly slower scale...cases even override genetically encoded patterning. Based upon trends in studies over the past several years...Chernet et al., 2016). There are many other light-based genetic tools for studying bioelectricity in vivo...desensitization in continuous light. ArcLight Q239 - A GEVI based on the fusion of the voltage sensing domain of a...

... Often, the choice for a fluorescent protein is based on the properties found in the literature or tabulated...is more sensible to compare fluorescent proteins based on their practical brightness in your experimental...mammalian cells this would be possible with CRISPR/Cas-based genome editing. Selecting a bright fluorescent protein...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene...system as well as the imaging method, we recommend basing your selection on a small-scale head-to-head comparative...

...the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started...with the death of Jesse Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong....cell) therapies. One may wonder why the first AAV-based gene therapy treatment for a genetic disease approved...forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive: clinical trials...for prevalent diseases - to name a few. CRISPR-based tools are also particularly well suited to be combined...and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina. Front Cell Dev Biol...

... sites in an orientation that results in 5’ four base overhangs after cutting. The destination vector ...IIS restriction enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the ...Single insert cloning efficiencies were measured based on the acquisition of antibiotic selectable markers...markers. The 5-, 12- and 24-assembly systems were based on the ability to correctly assemble a lacI/lacZ... over 50 years, prefers ligation of Watson-Crick base pair substrates, it demonstrates significant activity...cohesive end ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction conditions...

...transcription. Sometimes enhancers are located thousands of base pairs away from the gene they regulate, but are ...detected with chromosome conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). ...increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of...DNA that are far away from each other in terms of base pairs, but close together in terms of where they...dCas9 and gRNA expression. Additionally, CAPTURE 3C-based sequencing requires a large number of cells (~5x107...Han, J., Zhang, Z., & Wang, K. (2018). 3C and 3C-based techniques: the powerful tools for spatial genome...

...any plasmid-based experiment, untreated cells should be included as these provide the baseline/standard ...one thing should be present in every experiment-based investigation of a hypothesis: proper controls! ...the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...the abstract and follow an example of a plasmid-based experiment, delineate its requisite control plasmids...interpretation of the results. Typically, plasmid-based experiments employ transfection, negative, positive...completely. These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...

...homodimerization of FPs targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is ...random insertions can be generated with a transposon-based strategy (Sheridan, 2002). Avoiding protein over-expression...first express the fusion protein from a plasmid-based system to assess whether the chimera is still functional...verified and it can be compared to what is expected based on immunofluorescence or properties of the protein...independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097...Klarenbeek, Jeffrey, et al. "Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness...PMID: 30886412. Mastop, Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single...

...controls for antibody-based experiments Primary antibody specificity Antibody-based assays have their own...remains the same.    Figure 2: Microscopy-based antibody experiments should include a primary antibody....   Application-specific controls Some antibody-based applications have very specific controls, which ...you to detect dead or dying cells so that you can base your analysis on living cells only (for example,...some of the more critical controls for antibody-based experiments, it is by no means an exhaustive list... more information about antibodies and antibody-based experiments, check out these blog posts.       ...protocols elicited the expected response and have a baseline with which to compare the experimental group. ...

...PBMCs with episomal vectors Episomal vectors are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA...

...other forms of antibody-based (e.g., nanobodies, ScFVs) and non-antibody based affinity reagents (e.g....institutions have proposed a shift to recombinant DNA-based antibody technologies (Bradbury & Plückthun, 2015a...

...guides employed by Cas9 or Cpf1. These short, 24 base DNA guides may be more user-friendly, since they...working with small capacity viral vectors such as AAV. Base removal RNA-guided CRISPR enzymes cause double-stranded...unfavorable. In a natural context, it’s hypothesized that base removal helps prevent the invading genome from “...property could be an advantage. However, repair after base excision would alter the target sequence and preclude...

...one-size-fits-all approach. You will have to select a tool based on your project and not general opinion. I could... features and enables you to select the tool(s) based on your project needs. There are 8 major categories... is important for users wishing to select tools based on their comfort level with operating a computer...sequence”, “identifier”, and “load”. Trends: Sequence based input is by far the most common with most tools ...for the first time, it is best to choose a tool based on immediate needs and re-evaluate after several...have come across to offer classes of parameters based on the type of CRISPR experiment being pursued. ...implementation to be too slow for practical use. The R based package CRISPRseek offers great utility and coupled...