We narrowed to 695 results for: prot

...can also be fused to a protein of interest to track the autophagy of that protein. Find the cytosol-targeted...mRFP-GFP-LC3 Tandem Fluorescent Protein, but following translation, this fusion protein is cleaved to generate ... Addgene.org Fluorescent protein plasmids and resources Fluorescent protein guide: Biosensors ...biosensor that’s comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3...autophagy biosensor. Its resistance to lysosomal proteases is key to its pH-dependent fluorescence. In neutral...variant at Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy biosensor contains mRFP and GFP ...to the autophagy-related microtubule-associated proteins 1A/1B light chain 3B, or LC3. LC3 is a commonly...

... expresses the endogenous protein, since folding of the recombinant protein may differ from the native... have since adapted it for a host of different proteins and small molecules from a variety of sample types...versatility lies in the user’s ability to modify the protocol to best fit their specific needs. Herein, we’ll...antibodies and thus are not suitable for low abundance proteins. Because the method uses a single antibody, it... use the same secondary antibody for any ELISA protocol that uses primary antibodies of the same isotype... and a series of wash steps removes non-target proteins and molecules from the assay. The user then adds...Advantages Disadvantages Direct Fast and simple protocolReduced chance of cross-reactivity Higher level of...

...GO works by affecting protein translation of different reporter proteins. Protein translation requires ...the translation of any detectable protein, not just fluorescent proteins. Figure 4: Reporters in Gene...codon to initiate protein expression, it can be used for an array of detectable proteins including fluorescent...monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin... editing capability of the seven human APOBEC3 proteins, finding that APOBEC3A and APOBEC3bctd are the...reporter that is converted into BFP (blue fluorescent protein) upon CRISPR/Cas9 homology directed repair (HDR... a base-editing reporter. As the initiation of protein translation at the start codon ATG is universal...

...give rise to an adenine base editor. A massive protein engineering effort by Gaudelli et al. resulted ... restore antibiotic resistance. To find such a protein, they created a mutant library of E. coli tRNA ... ABE does not require any additional inhibitor protein like UGI in CBE. Base editors for the plant genome...based on the location of the ‘A’ relative to the protospacer adjacent motif (PAM). ABE7.10 can edit target...'A' if it lies in a small genomic window: at protospacer position 4-8 counting the ‘NGG’ PAM as 21-23 ...closer to the PAM, i.e., positioned at 8-10 in protospacer, ABE6.3 or ABE7.9 are better suited (Gaudelli...two Bsa1 sites to clone the single guide RNA (protospacer+scaffold) in a pol-III (OsU3) promoter driven...

...fluorophores to proteins, pioneers of this technique were able to directly detect protein/protein interactions... CFP-YFP was the first to be used to study protein-protein interactions and several other pairs have been..., Davidson MW. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells...FRET-based biosensors, and interactions between DNA and proteins. FRET, in theory, is an easy technique, but it...to studying an unknown interaction between two proteins, these parameters are difficult to overcome but...used to create your choice of fluorescent fusion protein with your gene of interest: Plasmid Color Expression...About FRET: Piston DW, Kremers GJ. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem...

...marks for safety.   Protein Delivery: Delivering reprogramming factors as protein is another way to avoid... vector-free Multiple transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free... inefficient Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods...are safer than integrating methods, with RNA and protein delivery considered the safest since there’s minimal...integration can occur. Okita et al describes a protocol for generating iPSCs by plasmid transfection where... following synthetic modifications are used to protect the RNAs from cellular defenses against ssRNA: ... exposing cells to exogenous genetic material. Proteins are delivered to cells by fusing them with peptides...

...Making proteins outside of a cell - cell free extracts Typically, if you want to make a protein – say ...DNA or transforming cells with green fluorescent protein, these activities are limited to those schools ...say green fluorescent protein – you first need the DNA sequence that encodes it. That DNA sequence then ...instructions it needs to produce that green fluorescent protein through transcription and translation. Unfortunately... live cells. The alternative is to express the protein in a cell-free manner. This is like taking an engine...systems contain all the parts needed to produce proteins without the need to handle a living, replicating...leaving them shelf-stable until water is added and protein production proceeds. FD-CF pellets are easy for...

...amino acids found in all proteins, so you can't distinguish the different proteins from each other. To distinguish...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...distinguish different target proteins in your flow analysis, you'll need to use fluorophores that attach...appropriate readout. A common method of labeling proteins is to fuse them with a fluorescent reporter, such...mCherry, through genetic modification. As your protein of interest will only occur in combination with... reporter's fluorescence. Another way to label proteins is through treating cells with fluorophore-conjugated...are completely different in nature (eGFP being a protein and FITC an organic molecule), their bleed is so...

...tool is the endoprotease Botulinum Neurotoxin B (BoNT/B), which cleaves the synaptic protein VAMP2, a protein...gas-filled protein structure that water-bound photosynthetic organisms use for buoyancy. These proteins allow...two fluorescent proteins and four repeats of a miRNA target site. One fluorescent protein (EBFP2) is expressed... protein essential for synaptic transmission. Cleavage of VAMP2 renders it inactive, thus inhibiting neurotransmitter... light-mediated? The researchers split the proteolytically active light chain of BoNT/B into two fragments...fragment with one part of an established pair of proteins (iLID and SspB) that undergo light-induced dimerization...function of microbes rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue...

...photoswitchable elements into many other types of proteins. Here, we’ll highlight a few recently-deposited...license. A naturally occurring microtubule-severing protein, Katanin is of course named for the samurai sword...monolayer of endothelial cells, showing cell protrusions and overlaps (arrows) after blue light illumination...optogenetic module to activate or recruit your protein of interest, maybe you want to inhibit it. Restricting...development and a knockout is not feasible or when the protein of interest supports a variety of processes in ...CIB1 clustering. Fuse either CRY2 or CIB1 to your protein of interest and express the other separately, then...generate CRY2-CIB1 oligomers that trap the fusion protein away from its usual location, preventing it from...

...Brainbow-1.0 construct contains three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP (membrane tethered...Brainbow-1.1 scales up the system to four fluorescent proteins, with recombination events specified by three ...principles remain the same. The first fluorescent protein in the construct, OFP (orange), is expressed when...invertible segments, each with two fluorescent protein coding sequences in opposite orientations. Cre-...Cre is removed from the system, the fluorescent protein whose coding sequence is closest to the promoter...fluorophores used are fluorescently distinct, their protein sequences are highly homologous, preventing design... First, they screened a variety of fluorescent proteins to find those with ideal characteristics (low ...

...antibody. Is this protein or protein region highly conserved across species? If the proteins are highly homologous...work very well for applications that denature a protein to its primary (linear) structure using heat or... an antibody in an assay for a native (folded) protein, such as immunoprecipitation, the epitope may not... trying to visualize the precise location of a protein in a tissue section then a directly-conjugated ...conjugates complex. If you are trying to visualize a protein in low abundance on a Western blot, a secondary...antibody that has been validated in the species your protein of interest comes from. If one is not available...different type of antibody and therefore each target protein. This can be accomplished by using a panel of primary...

...Photoactivatable Fluorescent Proteins Control Protein Expression with Cre-lox Control Protein Activity with GFP ... other kinases and proteins. Zhou et al suggest that more photoswitchable proteins could potentially be...transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to the next members of... by inserting pdDronpa domains into loops of a protein of interest. If you plan to try this, it’s important...important to check that the function of the protein and pdDronpa are preserved. References 1. Zhou, Xin...reporter plasmids Find photoactivatable fluorescent proteins in empty backbones in the Michael Davidson Collection...Photoswitchable Fluorescent Proteins ...

...chemically competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene, we use...using the protocols provided above, then you can find the subsequent transformation protocols below: Untergasser...chemically competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared ...streamlining the transformation step. Read on to find protocols and tips that you can use in your own lab. How...competent cells will work just fine.  A number of protocols for making competent cells are available online...can be transformed without heat shock. Detailed protocols are available via Zymo Research. In brief, we ... us less than 5 cents per transformation. This protocol has worked well for us for multiple E. coli strains...

...caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant protein rescue in living ...Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of human proteins that... his lab’s research is the mechanisms by which proteins assemble into functional multi-subunit macromolecular...rare diseases. Spontaneous mutations that change protein sequence affect arginine (Arg) amino acids more...function of even a small fraction of Arg-mutant proteins could improve health outcomes for millions of ...large number of other disease-linked Arg-mutant proteins both in yeast and human cells in order to determine...rescue of a variety of disease-linked Arg-mutant proteins in the lab, clinicians might consider prescribing...

...types of proteins: fiber, penton and hexon proteins. The viral genome DNA is further protected by histone-like...histone-like proteins forming the nucleocapsid. B) Three generations of adenoviral vectors: the first generation...helper adenovirus is required to provide the viral proteins in trans. The Gutless AdV have generated high ... systems are still being developed. A detailed protocol for production of Gutless AdV can be found in ...by looking at the ration of DNA (260nm) versus protein (280nm) absorbance. The ratio for the purified ... Enjoy! References 1. Luo, Jinyong, et al. "A protocol for rapid generation of recombinant adenoviruses...adenoviruses using the AdEasy system." Nature protocols 2.5 (2007): 1236. PubMed PMID: 17546019. 2. He TC. et ...

... process. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each ...VP64 fusion protein engineered with aptamers that bind to MS2 proteins. These MS2 proteins then recruit...simplicity of gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only...activation CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability, to bind...transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems...CRISPR activators is that it requires a fusion protein, rather than relying on a two-component system ...Gilbert LA, Qi LS, Weissman JS, Vale RD (2014) A Protein-Tagging System for Signal Amplification in Gene...

...It was a bit intimidating, but I had my lab’s protocols and some great co-workers to help me out. Even...writing AAV production tricks into the margins of my protocol. While these tips weren’t critical to the experiment...replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded intermediate into...single-stranded genome that's then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which ...HEK293 cells. Check out the Addgene AAV Production protocol for more details. Pro tips I’m not going to lie...Stable. NEB Stable competent cells lack RecA, a protein that aids in the homologous recombination of duplicated...enough AAV. Compared to the Addgene AAV Production protocol, I take a lazier approach for harvesting my AAV...

...yellow fluorescent protein mGold2 By Mike Lacy Get your sunglasses — these proteins are bright! Most current...post: Bright and photostable yellow fluorescent protein mGold2 Viral delivery of TnpB for plant genome ... current yellow fluorescent proteins tend to photobleach rapidly, which hinders their use for long-term...recently developed a pair of new yellow fluorescent proteins: mGold2s and mGold2t (Lee & Lai, et al., 2025)...or mCitrine, or if existing yellow fluorescent proteins just haven’t met your needs, consider trying mGold2s...2025). Bright and photostable yellow fluorescent proteins for extended imaging. Nature Communications, 16...production processes. Check out our step-by-step protocols for AAV production, purification, and titration...

...recapitulate the results of Gao et al 2016 in a letter to Protein & Cell. Update (August 3rd, 2017) THE ORIGINAL ...activity of the enzyme. In vivo, only nascent NgAgo protein (probably while it is being synthesized) can form...section asked basic questions about the NgAgo protocols used by respondents (transfection method, amount... to wait until someone else optimizes an NgAgo protocol for their favorite model system. The vast majority...trickiness of NgAgo is the inability of mature NgAgo protein to load an ssDNA guide. It is possible that, during...translation, only a small fraction of nascent NgAgo protein molecules manages to form a functional complex ...system. I encourage you to share your results and protocols with the research community if you've had success...