We narrowed to 695 results for: prot

...Activity of Protein Variants on a Large Scale Using Deep Mutational Scanning.” Nature Protocols 9.9 (2014... test how multiple amino acid changes affect a protein of interest’s function. This technique relies on...library that expresses all desired variants of a protein. Applying a selective pressure winnows the pool...the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed ... mutational scanning Hundreds of thousands of protein variants can be tested in one experiment. There...possibly discover unexpected epistatic changes in protein activity. See Natarajan et al for an example of...reaction. Below is a step-by-step overview of the protocol: Prepare ssDNA template: The wild-type plasmid...

...Resources on addgene.org Western Blot Protocol Western Blot Protocol Video ...Western blots are a great tool to identify a protein of interest in a complicated solution like cell ...— and what if you want to detect more than one protein in your sample? Or what if something weird happened...membrane, which is sturdier and retains the blotted protein better than a nitrocellulose membrane. If you have...probe for. Even using PVDF, some of your target protein will be removed from the membrane along with the...methods. If you are planning to detect multiple proteins through several rounds of stripping and reprobing...your signal by starting with the least-abundant protein and working toward the most abundant. If the abundance...

...published a protocol that would work for your system. Addgene has depositor submitted protocols and links...transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed gRNA...Single cells Cargo Nucleic acids or protein Nucleic acids or protein Throughput High Very low Efficiency...Do you want to create a fusion with a reporter protein such as GFP? All of these outcomes can be most ...deliver the components as DNA, or would mRNA or protein delivery be more suitable? Determine how you will...nucleofection, viral transduction or injection as either protein, RNA, or DNA. Finally, once you have identified...components, expression decreases as gRNA and Cas9 protein are degraded within the cell.   Delivery methods...

...the viral protein reverse transcriptase (RT). In the case of a natural virus, both the protein and the ...the fact that the virus must have its structural proteins expressed from a reverse-transcribed piece of ...gene therapy vector! For viral vectors, only the protein for reverse transcriptase (RT) is present in the...may seem like a mystery. But it’s simple. The RT protein is made by a separate set of instructions in the...components of the vector particle, some of the RT protein gets packaged inside of the vector particle, along...of the Central Dogma of Biology (gDNA → mRNA → protein): their roadmap includes RNA → DNA (Cooper & Temin...retroviral vectors can still direct the translation of proteins because they integrate this reverse-transcribed...

...identify protein and nucleic acid binding partners at numerous genomic loci through proteomics and the ...al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a genomic region...DNA-binding molecule, such as Zinc-finger or TAL proteins, can be used. The Fujii lab demonstrated this ...spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus. Figure...that do not use dCas9, require Zinc-finger or TAL proteins targeting a specific genomic locus, which can ... addition to the identification of DNA binding proteins, this method can characterize nucleic acid-nucleic...CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated with fragmented...

...capsid proteins are analyzed. The abundance of viral capsid proteins as a fraction of total protein present...electrophoresis. Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample...distributed to customers. Details about our production protocols, titering methods, and quality control are described...elements and an internal control of known titer (protocol modified from Lock et al., 2014.) Previous experience...vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC). ...by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties of the...relative stoichiometric ratios of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations...

...prime editing and found the protein La (a small RNA-binding exonuclease protection factor) promotes editing...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17] and Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct...the SARS-CoV-2 nucelocapsid protein, one of the virus’s most abundant proteins and a critical component ...SARS-CoV-2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein (Plasmid #141386-LV [+M]...transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt Adamson...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism...prime editing with an endogenous small RNA-binding protein. Nature. 2024 Apr 3. doi: https://doi.org/10.1038...

...viral proteins in human cells and used these viral “bait” proteins to fish for human “prey” proteins that...UCSF). Our approach is to discover key human proteins that can be targeted (hopefully by existing therapeutics...the virus. As a result, we identified 332 human proteins that the virus interacts with in cells. Because...Because viruses rely on the help of human proteins in their lifecycle, identifying these potential human helpers...better, more than half the drugs hitting these proteins are FDA-approved or in clinical trials or preclinical...understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T cells in our first...set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes an additional...

...two parts: 1) an activatable protein (the FAP - fluorogen activating protein - dL5** in this case), and ...you can do just that.  Photosensitizing dyes and proteins have been around for awhile (check out this review...recently developed a photosensitizer composed of the protein complex complex and the MG-2I-dL5** fluorogen that...few definitions. A photosensitizer is a dye or protein that absorbs light to produce reactive oxygen species...produced by photosensitizers inactivate target proteins or cells while having little effect on neighboring...fluorogen is not fluorescent until it binds its protein partner thus forming the full FAP-TAP complex (...suggests that it could be used with these fluorescent proteins. He et al also compared MG-2I-dL5** head-to-head...

...streak plate challenge. Chromoproteins are a subset of the fluorescent protein family that give off color...? Find the chromoprotein expressing plasmids here, and check out our streak plate protocol video on Youtube...Addgene.org Find fluorescent protein plasmids at Addgene Find chromoprotein plasmids at Addgene Check out...outcompeted the other species.  When he found the chromoprotein plasmids available from Addgene, he knew it ...color in ambient light. Expressing these chromoproteins in E. coli would make it easy to distinguish colonies...addition to streaking for single colonies, these chromoprotein-expressing strains are easily amenable to other...blog Read our other blog posts on fluorescent proteins Check out educational resources on the blog Resources...

...GFP-nanobody rather than a specific protein of interest. This fusion protein can then be co-expressed in essentially...stabilizes the TurboID-nanobody protein and allows TurboID to start labeling proteins in close proximity to the...Proximity-dependent labeling has become a powerful approach for proteomic mapping in both cell culture and whole organisms...approach has been hampered by the need to tag each protein of interest and, for in vivo work, generate a dedicated...zebrafish line expressing a GFP-tagged version of a protein of interest. The binding of the GFP nanobody to...the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The modularity of...including limitations of number of flourescent proteins that can be inserted into a cell without disrupting...

... 100 kD proteins or 20, 40, 60, and 80 kD proteins. Plasmids containing 150 or 250 kD proteins are also...expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins run as ladders...for multiplexed gene perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system for...to clone new constructs to label your favorite protein! While the target only requires a small epitope...doi: 10.1038/s41587-020-0600-6. The Penn State Protein Ladder  by: Rachel Leeson This simple, inexpensive...opportunity for lab members or students new to protein expression and purification. Figure 3: The...express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli, either individually (~20,000 lanes...

...domain-containing proteins may be broader regulators of Cas13d activity. This property makes WYL proteins an intriguing... near a putative effector nuclease. The Cas13d proteins they identified have little sequence similarity...HEPN-independent. These enzymes also do not require a protospacer flanking sequence, so you can target virtually...advantages, including Cas13d-specific regulatory proteins and high targeting efficiency, both of which are.... Yan et al.: WYL-domain-containing accessory protein WYL1 increases RspCas13d and EsCas13d cleavage ...The majority of Type VI-D loci contain accessory proteins with WYL domains (named for the three conserved...Biotechnologies found that RspCas13d accessory protein RspWYL1 increases both targeted and collateral ...

...Fluorescent Protein-Based Tools Learn Some of the Pitfalls When Using Fluorescent Proteins Control Protein Activity...Fluorescent Protein Plasmids Find Fluorescent Protein Kits Find Empty Backbones with Fluorescent Protein Tags...researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach is not...telomeric sequences, Chen et al. successfully labeled protein-coding genes with both intronic and exonic gRNAs... ortholog was fused to a different fluorescent protein to create three distinct colors. The specificity...two hairpin sequences, which recruit fluorescent proteins BFP, GFP, and RFP fused to the respective hairpin...better. Adding an additional hairpin/fluorescent protein combination, for example, with a far-red FP, would...

...with BioRender.com.   Opto-G-protein coupled receptors  G-protein coupled receptors (GPCR) regulate... Sensing G Protein-Coupled Receptor Signaling. In D. Niopek (Ed.), Photoswitching Proteins (Vol. 2173,...tools: light-gated ion channels, optogenetic G-protein coupled receptors (OptoXRs), and photoswitches....are many different opsin (natural and engineered proteins responsive to light in combination with retinal...concentration gradient, and of activation inhibitory (ArCH) proton pump passing ions against the concentration gradient...similar concept, except the ligand is attached to a protein binding partner or nanobody that targets the native...

...suitable antibody against your target protein, consider fusing a protein tag to the target and using an epitope-based...Frozen Tissue-V2 v2. protocols.io. https://doi.org/10.17504/protocols.io.bs68nhhw Im, K., Mareninov, S...antibodies available at Addgene Protocols for Immunocytochemistry Protocols for other antibody applications...with tissues), where an antibody binds a target protein in your sample, then a fluorescently labeled secondary...then you use a microscope to see where the target protein is (localization) and how much there is (quantification...quantification). But what if you’ve got several interesting protein targets and you want to know if they co-localize...the nuclei. (See Brewer et al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your...

...extracellular proteins with community-based validation. They also have plans to expand to other protein technologies...foundation to begin experimenting. Protocols.io, JoVe, and Bio-protocol all work to provide more descriptive...Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end) - Panel Alex...Researchers should be able to follow one another’s protocols and use one another’s reagents with the confidence...that aren’t always mentioned in static written protocols. It is therefore a bit ridiculous to expect that...to share in-depth information about reagents, protocols, analysis tools, and more. Being that this is ...Steve Almo’s talk about work at the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also...

...lines. Identifying less toxic envelope proteins or envelope proteins that target specific tissues are areas...that's gag, pol, and env), the isoforms of these proteins, as well as the viral long terminal repeats (LTRs... gene of interest. Consequently, the levels of protein expression tends to be much lower than those seen...including 293 were immortalized with adenoviral protein E1A, which has been shown to repress expression... 5'LTR, which requires the presence of the Tat protein to work, while 3rd-generation transfer plasmids... 5'LTR removes the requirement for the HIV Tat protein, thus decreasing the probability of creating replication-competent...envelope plasmid and a packaging plasmid encoding HIV proteins gag, pol, rev, and tat. 3rd generation systems...

...replaced the protein-based DNA-binding domain with a single-guide RNA sequence.  The Cas proteins, just like...catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the prototypical base editor enzymes....CRISPR-Cas9 system. Nature Protocols 8:2281–2308 . https://doi.org/10.1038/nprot.2013.143 Seligman LM (2002...Maclyn McCarty’s experiment debunked the myth that protein is the carrier of genetic information and led to...enzymes, meganucleases, were discovered. These proteins are characterized by large DNA recognition sites...Era: Post CRISPR  CRISPR The strategy of using a protein to recognize DNA sequences is not ideal. For the...target new genomic sites. The discovery of the Cas proteins in 2005 was a major breakthrough in the world ...

...determine how Protein A and Protein B interact. You read extensive research on the two proteins and come up... an ideal antibody against Protein A and an ideal antibody against Protein B. The antibodies are both ...to detect Protein A and green fluorophore-tagged anti-mouse IgG2a secondary to detect Protein B and successfully... anti-Protein A antibody and an anti-mouse IgG2a secondary antibody to bind to the anti-Protein B antibody...are rat IgG1, then you could convert your anti-Protein B antibody to a mouse IgG2a Fc. The resulting chimeric...successfully visualize both proteins.     Figure 2: Two primary antibodies with the same isotype...