...allows you to ask questions like: “Where does my protein of interest live within a cell,” “Does this disease...where they bind to their targets, most commonly proteins. You can then use fluorescent microscopy to look... multiplexing is that you can ask how multiple proteins relate to one another in the same sample. For ...dozens of products that all claim to target your protein of interest. Thankfully, there are a number of ...experiment.    References and resources References Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction...allowing one to “directly” visualize their target. In contrast, indirect IF requires two antibodies: a primary...

... Backbones Fluorescent Protein Guide: Empty Backbones More Fluorescent Protein Resources: FRET Biosensors...fusion proteins with your gene of interest. These are organized By Color of the fluorescent protein and ...The original green fluorescent protein (GFP) was cloned in 1992 ( Prasher et al., Gene, 1992 ), and since...create fusion proteins with your gene of interest. Special... Special Collections Fluorescent Protein Guide Empty ...Imaging Blog: Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs Blog: When is a Monomer not...not a Monomer? Background Fluorescent proteins are genetically encoded tools that are used extensively...

...bacterial strain that is deficient in recombinase protein RecA can reduce the frequency of multimerization...large inserts are often dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147...microbiolspec.PLAS-0025-2014. PMID: 26104344. Fishel RA, James AA, Kolodner R. recA-independent general genetic...

...approaches to study protein interactions is the pull-down assay, wherein a protein of interest can be ...IP/MS, a target protein is first immunoprecipitated - along with its associated protein complexes - from...using antibodies against the target protein. The captured protein complexes are then analysed by mass ...interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest...target protein. Both IP/MS and ChIP-seq methods provide a wealth of information about how protein networks...pre-validated antibody for each target protein. To study 20,000 human proteins, this would be time-consuming, ...tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins can be...

...reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted...sites. Alternatively, if you want to make your protein non-functional, you may simply introduce a frame-shift...PMID: 8470801. PubMed PMID: 8470801. 3. Saiki, R. K., et al. "Primer-directed enzymatic amplification ...you need a high-fidelity polymerase. There are many high fidelty polymerases on the market; you need one...a DNA polymerase that produces blunt-ended PCR products (e.g. Phusion, Pfu, and Vent polymerases). Note...amplified fairly easily by simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust...plasmid template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired ...

...Promoter Expression System PI 8394 p209 pCMV-cre-K Cre-K CMV Mammalian Green 8395 p210 pCMV-CREM CREM CMV...specific system for controlling gene expression. The protein Cre recombinase recognizes 34 bp loxP sites, and...fusion; for the production of VLPs loaded with CRE protein CMV Mammalian Ohlmann 120279 014LP-pAAV-ANF-657...stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons...Zeng 61573 Rasgrf2-2A-dCre targeting vector DHFR-destabilized Cre; targeting vector for Rasgrf2 Mammalian...backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR Cre, luciferase, and sgRNA expression EFS AAV Zhang 60229 AAV...Retroviral Luikart 67503 pF CAG luc-EGFP-cre puro Luciferase - EGFP- Cre fusion CAG Mammalian Stringer 68448...

...naturally-occurring Acr (Anti-CRISPR) proteins can help control CRISPR activity. Acr proteins have evolved in phages ...; or inducing degradation of the Cas9 protein. Most Acr proteins are specific to a particular type of ...and antibody-activator effector proteins; long-term imaging of proteins in living cells (Figure 9B) dCas9...with different fluorescent proteins or by fusing the gRNAs to orthogonal protein-interacting RNA aptamers...applicability of base editing through protein engineering and protein delivery. Nature Communications . 8... by anti-CRISPR proteins. Nature , 526 (7571), 136–139. PMID: 26416740 Bravo, J. P. K., Liu, M., Hibshman...see Cas3 for more details), while type V-K systems use a type V-K Cas effector, Cas12k. No matter the system...

...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...probably due to the presence of nucleosome or other proteins. You can detect base editing as early as 48-72...29807545. PubMed Central PMCID: PMC5972399. 12. Molla, K., Shih, J. & Yang, Y. Multiplex CRISPR-mediated base...inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair...

...Hirabayashi T, Fujimoto A, Mimura K, English JG, Liu J, Inoue K, Kumata K, Seki C, Ono M, Shimojo M, Zhang...transmembrane proteins that bind a specific ligand. Ligand binding then activates G-proteins to modulate...signaling protein that the receptors couple to: Gq-DREADDs signal through the Gαq/11 G-protein and activate...Na + , K + influx Neuronal activation Björefeldt et al., 2024 LMO11 GeNL_SS ChRmine CFz Na + , K + influx...17360345 (Link opens in a new window) Berglund K, Clissold K, Li HE, Wen L, Park SY, Gleixner J, Klein ME...DREADDs), like RASSLs, are based on engineered G-protein coupled receptors, but unlike RASSLs, show insensitivity...Chemogenetic Receptors: RASSLs The first chemogenetic receptors were based on G-protein coupled receptors, ...

...other small base pair changes, and the addition of protein tags. It also has the flexibility to modify the...: 1) use a strain of bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos... not the amino acid sequence of the translated protein.  These changes can be 5’ or 3’ of the desired ... detail below).  Controlling expression of Red proteins is critical for minimizing the toxic effects of...10811905.PubMed Central PMCID: PMC18544. 3. Murphy, K. 2016. λ Recombination and Recombineering, EcoSal ...105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability for generating dsDNA...nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase to generate insert ssDNA Point mutations or...

...repair proteins fused to CGBEs The David Liu lab also experimented with adding different proteins to the... binding motif protein X-linked (RBMX) (Koblan et al., 2021). Several of these proteins had additive effects... the DNA repair protein rXRCC1, or the DNA binding and lyase domain of DNA polymerase β (PB) (Chen et ...UdgX, they found three proteins whose fusion improved the CGBE: DNA polymerase D2 (POLD2), exonuclease... cytosine transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the Wei Leong...improving C → G editing. Instead of fusing DNA repair proteins to the base editor, they added the pioneer factor...with CRISPR-Cas9-directed base excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org...

...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... exposing cells to exogenous genetic material. Proteins are delivered to cells by fusing them with peptides...Kim et al, cells were exposed to reprogramming proteins for 8 hours a week for 6 weeks, and still required...and it’s difficult to purify the reprogramming proteins needed to perform these experiments. Assays for...

...in a direct or unbiased way at the DNA, RNA, or protein level. The “correct” choice of tool requires careful...unmatched—unlike CRISPR experiments that require exogenous protein, successful induction of RNAi only requires the...scored. However, determining the transcript or protein abundance of your specific target required physical...Bush J, McCormack M, Sheen J (2013) Comprehensive Protein-Based Artificial MicroRNA Screens for Effective... M, Grandi P, Jakob P, Michon A-M, Sun H, Tessmer K, Bürckstümmer T, Bantscheff M, Steinmetz LM, Drewes... a targeting sequence for the adenine phosphoribosyltransferase (APT) gene, which is conserved in many...

...the same page in terms of the vocabulary--DNA, protein, plasmid, gene, etc. Though I talked about CRISPR...teach  5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill levels... I came up with an analogy that would help them grasp some of the key concepts (Fig. 2). One thing I will...

... be used as a proxy measure for differences in protein expression. (If you're just getting started, check... amplicons. Image credits (respectively): Zuzanna K. Filutowska; Selma, 2019; Trampuz, 2003.   You...doi.org/10.1038/s41598-017-02217-xTaylor, SC; Nadeau, K; Abbasi, M; Lachance, C; Nguyen, M; Fenrich, J. The...References and Resources  References Biassoni R; Raso A. (eds.), Quantitative Real-Time PCR: Methods and...

...release proteins from cellular membranes using light. light-induced protein dissociation using protein A fragments...photoreceptors to create protein systems controlled by light. These “photoswitchable” proteins offer exquisite ...of a protein of interest. A genetically-encoded nuclear Cry2 fusion can thus direct a CIB1-protein fusion...Inhibits protein function by reversibly sequestering targets into large, optically assembled protein architectures...transcription using engineered zinc finger proteins, using proteins GIGANTEA (GI) and the light oxygen voltage...pair to the target protein, and the other to the membrane where the target protein is to be sequestered...associate and cage a protein in the dark, while dissociate and activate the protein in the light ~400 (...

...components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting module with...with the crRNA, (3) three transposase proteins, and (4) the donor DNA. By using a multi-spacer CRISPR array...Zalatan’s lab used an approach to identify known proteins involved in the activation of transcription including...including transcription factors, phage proteins, and RNAP components (Dong et al., 2018). They found a...dCas9 variant fused to bacterial enhancer binding protein required for the activation of sigma-54 type promoters...glycosylase inhibitor and degradation tag on the fusion protein to limit the activity of the system as a whole.... Rep 6: . https://doi.org/10.1038/srep19452 Selle K, Fletcher JR, Tuson H, Schmitt DS, McMillan L, Vridhambal...

...complexes to study neural function. Often the reporter proteins associated with these complexes are so robustly...vectors: packaging capacity and the role of rep proteins in vector purity." Human gene therapy 18.2 (2007... 1248. PubMed PMID: 11509958. 8. Ferrari, Forrest K., et al. "Second-strand synthesis is a rate-limiting...Surprisingly, little research has compared and contrasted optogenetic and chemogenetic receptors delivered...

..., Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, Nguyen K, van der List D, Murray KD, Engebrecht...exciting for scientists working to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling...

... Collections Fluorescent Proteins Fluorescent Biosensors Fluorescent Proteins: Biosensors Metal Ions Small...-Aequorin Protein) for imaging of Ca+ dynamics in endoplasmic reticulum GFP-Aequorin Protein Sensor for... Red fluorescent protein-based cAMP indicator (Pink Flamindo) Red fluorescent protein-based cAMP indicator...of Redox-sensitive protein sensors (roGFP) Lifetime imaging of a fluorescent protein sensor reveals surprising... (Constitutive or Cre-dependent) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature...Service: AAV Biosensors Fluorescent biosensors are proteins designed so that their fluorescent properties ...in signal of a circularly permuted fluorescent protein (cpFP) or Förster resonance energy transfer (FRET...