We narrowed to 1,405 results for: ins

...                           California Institute of Technology             Johns Hopkins University                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...for tagged and untagged expression of SARS-CoV-2 proteins in bacteria, yeast, and mammalian cells. We teamed...from the Michael J. Fox Foundation are used in Parkinson’s research.  The extensive resource from the Structural... determine the three dimensional structures of proteins.  The RESOLUTE Consortium collection enables research... collection, a ‘One-Stop Shop’ for Fluorescent Proteins, and plasmids from several labs at Florida State...

...own machinery to replicate the viral genome and proteins. This is, for the intrepid researcher looking ...Virus structure While all viruses have a genome inside a protein shell known as a capsid, some also have...can either follow central dogma (gDNA to RNA to proteins) or require an intermediary DNA replication step...transcribed into mRNA, directly translated into proteins, or, in the case of retroviruses, reverse transcribe...transcribed into mRNA before being translated into proteins. RNA viruses replicate their genomes using a variety...egg sac. (Okay, some enveloped viruses bud out, instead of lysing, but they do generally still kill the...replicative ability. The remaining particle, which retains the protein coat (and envelope), has a “gutted”...

...pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane...update our iGluSnFR post! Here, we look at the origins of the system and explore advances that have happened...happened since our original 2018 blog post. The origins of iGluSnFR   iGluSnFR was developed in 2013 by...an increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing...localization through different C-terminal anchoring domains The original iGluSnFR in the pMinDisplay backbone...introduce iGluSnFR3 in two additional anchoring domains – GPI and SGZ. Fig: 4 Sequence schematics...Fluorescent Protein Biosensors Browse All Fluorescent Proteins Find AAV Tools References Marvin, Jonathan S....

...transposon to stably integrate a reporter gene that contains a puromycin resistance gene followed by the target...the desired genome edit:  A donor plasmid that contains a puromycin resistance gene linked to an in-frame... when transfecting your cells.  A plasmid that contains Cas9. The Madison lab also created an inducible... labor-intensive, require multiple fluorescent proteins, and aren’t that efficient. To overcome these ...named GEmCherry1, due to the “genomic fragment inserted into mCherry”. However this one mutated methionine...GEmCherry2 work? The premise is that the genomic region inserted into the N-terminus of mCherry can be targeted...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of...

...constitutively expressed proteins while tet-on turns on constitutively silenced proteins. Addition of tetracycline...degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron tags, just ... to temporarily reduce expression of essential proteins (when knockouts aren’t viable). siRNA or RNAi ...gene of interest. It will inhibit transcription instead of introducing a DNA double strand break. Transient...transgene vector. Degron tags Tagging relevant proteins with a degron tag can provide inducible protein... easily implemented for exogenously introduced proteins, as many of them just need a special promoter ...

...new Cas enzymes on the block? The Cas family of proteins has many useful genome engineering enzymes, each...other Cas enzymes is its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean...Unlike many other Cas enzymes, Cas14 targets ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity -...nuclease, however, unlike Cas9, it targets RNA instead of DNA. This makes it particularly useful in generating...composed of the fusion of several Cas7 and Cas11 domains. Cas7-11 cuts ssRNA and is capable of pre-crRNA...   Table 1: Fast facts on Cas family proteins and genome editing abilities   Choosing a...adaptable Cas currently available, the other Cas proteins should not be overlooked. The unique features ...

...enzymes that degrade RNA, are especially hardy proteins that are found in and on everything, including...solution and spun down the homogenate to remove the insoluble material. Then, the homogenate was loaded onto... hours to separate the intact RNA from DNA and proteins. Although very effective at isolating total RNA...RNA could be effectively separated from DNA and proteins by a simple extraction protocol with guanidinium...in a guanidinium thiocyanate solution. However, instead of RNA separation using cesium-chloride gradients...in the top, aqueous layer, while DNA and other proteins are retained in the interphase and bottom, organic...into one step. After homogenization in TRIzol®, insoluble material is removed via centrifugation and the...

...detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield, 2014). Traditionally,...Therefore, researchers from over 100 scientific institutions have proposed a shift to recombinant DNA-based...expression. If starting from a protein sample (for instance, affinity purified antibodies from a patient’s...the line and subsequent cloning of the antibody chains (Crosnier et al., 2010; Andrews et al., 2019). ...well in animals because of similarities to host proteins. After the antibody of interest has been cloned...non-antibody based affinity reagents (e.g., monobodies, DARPins) (Helma et al., 2015).    The Many Benefits of...and sequences for antibodies are not shared. For instance, through open plasmid sharing, the CRISPR community...

...add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked the students...perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent protein family...off color in ambient light. Expressing these chromoproteins in E. coli would make it easy to distinguish...single colonies, these chromoprotein-expressing strains are easily amenable to other experiments to teach...have the students transform them into different strains of E. coli,” says Westenberg. During the transformation... how different genetic backgrounds of the host strains affect transformation efficiency.  Aside from the...blog Read our other blog posts on fluorescent proteins Check out educational resources on the blog Resources...

...modulation of endogenously and exogenously expressed proteins. Here we will review what advantages degron tags... the tagged protein. Degrons are destabilizing domains which will be identified by various enzymes and...manipulate endogenously and exogenously expressed proteins. Thirdly, degrons and their drugs are titratable...not amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag self-...Promega as an affinity tag for visualization of proteins of interest. A fluorescent Halo ligand can be ... molecules which target E3 ligases to specific proteins by simultaneously binding both their target protein...their effectiveness with over a dozen different proteins. In the end, you can’t predict with absolute certainty...

...into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified...systems. Insect expression systems Insect cells can also be used to produce complex eukaryotic proteins with...Researches can visualize proteins in vivo by tagging them with fluorescent proteins to study localization...localization or purify proteins to study their structure, interactions and functions. Proteins can also be purified... to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively easily...are two types of insect expression systems; baculovirus infected and non-lytic insect cells. Baculovirus...genes integrated into the insect cell genome. Both of these types of insect expression systems can be ...

...Harrach, 2014). Given that the early genes encode proteins essential to initiating the whole process – such...used to house a recombinant sequence (transgene) instead.    Although the late genes are usually present...absent, and the late genes (and their structural proteins, which would encapsidate new virions) are not ... is transient due to the fact that AdV surface proteins (especially fiber’s knob domain, and hexon), as... as well as other intracellularly-exposed proteins and downstream cellular events, all recruit strong ...sense of what AdVs are about – and we hope that it inspires further use of existing vectors, as well as research...resources References Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., & Hermiston, T. W. (2003). Multigene expression...

...now available. In this post, we’ll review the inspiration behind ExM, how it works, and some of the limitations...most SRM techniques also require specialized instruments and technical expertise, limiting their accessibility...a straightforward answer to the space problem. Instead of imaging smaller, what if we just made the sample...chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016...adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al. 2019). Perhaps...et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including...Saad O, Bewersdorf J (2020) Light microscopy of proteins in their ultrastructural context. Nat Commun 11...

...       Insert-specific primers: Insert-specific primers are designed to anneal to an insert-specific... your insert or if the insert is in your plasmid Have to make a new primer pair for each insert Backbone-Specific...the insert, insert orientation, and the sequences of the junctions between the plasmid and insert DNA....negative clone without the insert. This type of primer pair can tell you if the insert is the correct size and...orientation of the insert. Orientation-specific primers: If you need information about insert orientation,...of the insert. One member of this type of primer pair anneals to a sequence flanking the insert and one...anneals to the insert. A simple way to create this type of primer pair is to mix-and-match insert-specific ...

...reporters as well as secretory proteins or proteins targeted to membrane sub-domains. It has also been used to... reporter mRNA, which contains the tethering sites for the fluorescent proteins and an inducible promoter...and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins that are bound...the nucleus (thanks to a NLS on these proteins). The RFP proteins, however, remain bound to the mRNA since...the Wyss Institute have extended the transactivating capabilities of CRISPR/dCas9-fusion proteins and have...engineered hook proteins fused to a streptavidin core that are able to retain reporter proteins fused to the...possible protein domains containing these properties, they generated monoclonal antibodies against them, fused...

...next-generation sequencing (NGS). However, Sanger sequencing remains useful for small sample sizes and shorter regions...occasionally still use Sanger sequencing to confirm an insert that is repetitive or otherwise hard to sequence...time-consuming, depending on the library prep and instrumentation used.  Here at Addgene, we sequence every plasmid...high-throughput compatibility. However, there are some instances where we use long-read sequencing or Sanger sequencing...DNA or RNA is pulled through the pore via motor proteins. When each nucleotide passes through the nanopore...nanopore, the electrical current recorded by the instrument changes. Base-calling software can then identify...SPAdes, Shovill, apc, and NOVOPlasty. The pipeline contains three de novo assemblers, so if one fails to generate...

... In this iteration, the size of the lentiviral insert was minimized to reduce recombination and avoid...transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt Adamson...nucelocapsid protein, one of the virus’s most abundant proteins and a critical component for viral genome packaging...-LV [+M], negative control) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid Protein antibodies...middle panel). Samples were also immunoblotted against vinculin as a loading control (right panel). Image... of ssAAV, a targeting sequence of miR124T was inserted into the vector to silence transgene expression...editing, and delivery of disease-relevant large proteins. They demonstrated REVeRT in various contexts ...

...of the viral capsid proteins are analyzed. The abundance of viral capsid proteins as a fraction of total...relative stoichiometric ratios of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations...subjected to gel electrophoresis. Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total.... Three to five days later, cell cultures are inspected for sterility. Transducibility Some viral vectors...virus from Addgene, read our detailed recipient instructions for next steps. Plan an experiment using our...plasmid in the viral preparation by performing PCR against unique regions of the transfer plasmid.   Lentivirus...

... open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible... allow your insert to be placed into the backbone in the proper orientation. For instance, if you were...enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert can then ...used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification ...separate your backbone away from any inserts cut out of it and your new insert from any overhangs cut off of ...these stains see the Bitesize Bio Blog and their associated manufacturers websites. These stains require...Your Insert into Your Vector In the ligation step, you mix your purified, cut backbone and insert in a...

...fused to two proteins, here proteins Y and Z, that are known to interact. When  proteins Y and Z interact...interacting proteins; nearby proteins are biotinylated when and where the two bait proteins interact, but...interact or from proteins that are just in the proximity of the bait proteins. Instead of pinpointing specific...restored, and nearby proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed in...activity is restored, and nearby proteins are labeled with biotin. When proteins Y and Z part ways, BirA* biotinylation...the biotin labeling remains even if the interaction with the BirA* fused bait proteins ceases. Lastly, Split-BioID...provides a candidate list of proteins that are part of the bait proteins’ interactome. Further characterization...