...the folded protein (green region). Methanol dehydrates the protein, which can disrupt protein structure...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples... (as the name suggests) cross-linking cellular proteins. Cross-linking is a chemical reaction that covalently... polymerize and lose its ability to cross-link proteins. As such, you will get the best results if you...solvents can be quite harsh - lipids and soluble proteins can be lost during fixation with these chemicals...these chemicals can also alter the structure of proteins, which could disrupt your target epitope (Fig....antibody binding. Formaldehyde cross-links the protein (red lines), which maintains structural epitopes...

...detection because they produce very little or no viral protein. To solve this problem, two strategies have been... HIV so that infected cells will produce viral proteins and die, either through cytotoxicity or the immune...expression. In cell lines that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating ...However, this approach could permit some viral protein production, so researchers are also interested ...antiviral therapies target HIV-1 at the level of protein structure, CRISPR gRNAs require DNA sequence-specific...Kaminski R, Fagan PR, Putatunda R, Young WB, Khalili K, Hu W. CRISPR/gRNA-directed synergistic activation...Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K. Elimination of HIV-1 Genomes from Human T-lymphoid...

...specifically curated for protein expression in K. phaffii with special attention to protein modifications which...successfully for protein localization and transport studies in HEK cells, recombinant protein purification...used to optimize protein secretion and expression in the biotech workhorse yeast — K. phaffii (formerly...great option for those interested in recombinant protein purification and metabolic engineering. If instead...library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent option (Iverson...compatible with E. coli and/or cell-free systems. Protein expression in yeast MoClo is also for eukaryotes...formerly known as Pichia pastoris). If your goal is protein isolation, then the MoClo Pichia Kit will get you...

...CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create...composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1...form a functional protein due to being fused with high-affinity interacting proteins Coh2 and DocS, respectively...chimeric protein consisting of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa... saCas9 protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are a highly...photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa) (Kawano et al.,... Nihongaki et al., 2015 paCas9 Magnet proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9...

...fluorescent proteins in the nervous system. Livet J, Weissman TA, Kang H, Draft RW, Bennis RA, Sanes JR,...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...

...few years, scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple,...has adapted natural RNase activity of the Cas13 protein to develop and optimize the method termed Specific...endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA 22:318–329 . https://doi.org/10.1261/...S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F (2016) C2c2...Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan C-Y, Guevara ...Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV (2015) Discovery and Functional...sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-...

...lymphocyte receptor signaling. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. 1998 ...170(5):956-972.e23 Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated...Epigenome CUT&RUN: An Improved Method for Studying Protein-DNA Interactions CRISPRainbow and Genome Visualization... NEAT1 RNA is essential for the structure of paraspeckles. Clemson CM, Hutchinson JN, Sara SA, Ensminger...

...mini counterpart label nearby proteins with biotin. The biotinylated proteins can then be isolated and identified...lysosomal-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase, ChAT) neurons. ...for Glypicans DNA editing with STITCHR Dynamic protein labeling with TurboCas Enhancer-AAV viral preps...our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression and AAV...therapeutic potential. Our partners at the Institute for Protein Innovation (IPI) recently released a set of highly-specific...antibodies against three of the six human glypican proteins (GPC1, GPC3, and GPC4). All IPI antibodies undergo...structure. Glypicans are proteoglycans composed of a protein core, 2–4 glycosaminoglycans (GAG), and are (typically...

...many of the specifics - promoters used, precise protein sequences or domains, and methods of delivery -... at CRISPR. References Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing ...10.1128/mmbr.67.1.16-37.2003 Khatodia S, Bhatotia K, Passricha N, Khurana SMP, Tuteja N (2016) The CRISPR...TTS, Sweeney CJ, Koman VB, Wong MH, Bohmert-Tatarev K, Snell KD, Seo JS, Chua N-H, Strano MS (2019) Chloroplast-selective...Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y-G, Zhao K (2016) Enhanced Rice Blast Resistance by CRISPR/Cas9...editing technologies especially in human cells. In contrast to homology directed repair, these methods do ...

...tissues, eliminating the need to deliver the Cas protein altogether. Platforming AAV Interest in interrogating...https://doi.org/10.1038/s41596-022-00700-y Hanlon, K. S., Kleinstiver, B. P., Garcia, S. P., Zaborowski...Chamberlain, J. S., Corey, D. P., Aryee, M. J., Joung, J. K., Breakefield, X. O., Maguire, C. A., & György, B....Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop, J. J., & Kampmann, M. (2023). CRISPR screening...lentiviral vectors being packaged using VSV-G. In contrast, AAV can be packaged in a variety of capsids. ...

...which “bait” proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with...2015. PubMed PMID: 26479688. RAS Pathway Clone Collection 2.0 Ras proteins are small GTPases involved in...central role Ras proteins play in cell signaling. Understanding how molecules in the Ras signaling pathway...method for identifying protein-protein associations. This method uses a bait protein fused to a biotin ligase...PrM N and C   Detecting protein-protein interactions with DULIP Proteins rarely act alone. Molecular...the NCI RAS Initiative, led by Dominic Esposito, has recently released a collection of 360 Ras pathway...ligase to biotinylate proximate proteins in the cell. These candidate proteins can be enriched via biotin ...

...modified reprogramming factor cocktail, and viral protein EBNA-1. Methods for reprogramming hiPSCs from ...EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences are necessary and... of the vectors in human cells [8]. The EBNA-1 protein recognizes OriP and induces amplification of the...transiently. The transiently expressed EBNA-1 increases protein expression from the other episomal plasmids. When...PubMed PMID: 14564000. 5. Seki, T., S. Yuasa, and K. Fukuda, Generation of induced pluripotent stem cells...29(5): p. 547-57. PubMed PMID: 25228828. 7. Okita, K., et al., An efficient nonviral method to generate...is easily and routinely drawn from patients in contrast to the process of collecting fibroblasts, which...

...covalent modifications to DNA or histones, the proteins that help package DNA, were associated with gene...With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier could be targeted ...cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways work ...NLS-Sa dCas9-NLS-LSD1. LSD1 is a particularly large protein, and this fusion allows for packaging into viral...exhibits increased PAM flexibility, and this fusion protein allows for LSD1 targeting to a wider set of genomic...methylation in vivo using an engineered dCas9-MQ1 fusion protein. Nature Communications 8: . https://doi.org/10.1038...endogenous genes using programmable TALE-TET1 fusion proteins. Nature Biotechnology 31:1137–1142 . https://doi.org...

...available to assess protein-protein interactions. One popular approach is to fuse a protein of interest to ...split fluorescent protein (FP) and measure the signal produced when the candidate proteins’ interaction brings... Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein A and B interact...Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R, Jiang F, Xie J,... and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. Nat Commun...efficient, and robust method for detecting protein-protein interactions in vivo. Blakeley BD, Chapman ...

... Addgene Blog Study Protein-DNA Interactions with CUT&RUN Study Protein-Protein Interactions with Split-BioID...like DNA and protein get all the attention.There are numerous ways to detect DNA-protein interactions ...CHIP-seq, FAIRE-seq, Cut & Run) and to detect protein-protein interactions (yeast-two hybrid, Co-IP, BioID...you want to study RNA-protein interactions? The characterization of RNA-protein interactions has lagged...non-physiological RNA-protein interactions due to sample processing. Require prior knowledge that the protein of interest...immunoprecipitation (CLIP) identify RNA-protein interactions by cross-linking RNAs and proteins with UV light, followed...antibody pulldown of a protein of interest, much like using ChIP for studying protein and DNA interactions...

...test varies in the sequence of steps and the Cas protein used. Here, all the components needed for the reaction...Assay (FELUDA) is a technique that uses FnCas9, a protein that is highly sensitive to the presence of mismatches...Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan C-Y, Guevara ...C., Braig, F., Lach, R. S., Baxter, N. J., Kosik, K. S., Arias, C., Acosta-Alvear, D., & Wilson, M. Z.... sample and subject to isothermal recombinase polymerase amplification (RPA) using a commercially available...CRISPR/Cas12a (AIOD-CRISPR) assay – for rapid, ultrasensitive and visual detection of novel coronavirus SARS-CoV...reaction also contains SSB, strand displacement DNA polymerase, recombinase, and the ssDNA-FQ reporters that...

... Resources Kulanayake, S., Tikoo, S. K. Adenovirus Core Proteins: Structure and Function. Viruses. 2021...vesicular somatitis virus G glycoprotein (VSVG). VSVG is a broad tropism envelope protein that can also be pseudotyped...interact with the host. This is the role of Gag proteins: they contain the matrix, capsid, and nucleocapsid... Viral packaging and entry The viral packaging proteins for AAV are rep and cap. Rep is required for viral...AAVS1 locus in humans. Cap is a structural capsid protein that helps with virion assembly and particle release...discussed. LXs/Iva2 are primarily capsid assembly proteins required for packaging of the virus into particles...The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase II promoter and terminator regions and are slightly...

...the name "G-protein coupled receptor" into two parts: the receptor itself and the G-proteins it associates...Takeda et al., 2002). G proteins: The receptor is coupled with heterotrimeric G proteins on the intracellular... of the heterotrimeric G protein complex that can be used to detect G protein activation via bioluminescence... catalyzes the biotinylation of proteins near the GPCR. These proteins are then identified with mass spec...besides being one of the five senses? GPCRs or G-protein-coupled receptors (GPCRs)! Aside from these three...recognizes the appropriate ligand to activate the G proteins. It is estimated that there are approximately ...intracellular side of the cell membrane. Heterotrimeric G proteins are important molecular switches in signal transduction...

...binding with Rep proteins and mutations in the trs site reduce Rep nicking. Since Rep proteins are important...Shalik, I. K., & Yudkin, D. V. (2023). AAV- based vector improvements unrelated to capsid protein modification...with the viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication...resolution site that serves as the target site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type...elements of AAV and rAAV vectors, modifications can drastically affect gene transfer efficiency and transgene...

...self-assembling protein (SAP) such as a bacteriophage shell protein or a bacterial microcompartment protein. The ...fluorescent proteins into one particle within the cell, the lab fused the red fluorescent protein (RFP) to... (DCDC), relies on the assembly of fluorescent proteins into one bright particle that segregates into ...PubMed Central PMCID: PMC6467691. Takahashi, Melissa K., et al. "A low-cost paper-based synthetic biology...systems in bee gut microbiome species such as Snodgrassella alvi and Gilliamella apicoli. The plasmids could...

...necessary to visualize expression of fluorescent proteins, for example, as many viral particles must infect...infect a cell to achieve visible expression of the protein. However, expression of receptors may not need ...In the case of channelrhodopsins, fewer channel proteins are required for the transduced cell to become...become light responsive than fluorescent proteins required to get a good fluorescent signal. It may not be...to improve AAV production.     References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated... by an anterograde AAV was shown to drop off drastically with lower titers. As you can see in figure 2...

...the viral protein reverse transcriptase (RT). In the case of a natural virus, both the protein and the ...while missing the polymerase gene may seem like a mystery. But it’s simple. The RT protein is made by a separate... there is a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which...two strands of viral RNA and of the viral RT protein (Faras et al., 1973). Interestingly, retroviruses ...from step 2 will be integrated by the viral integrase protein (IN) during step 3 (King et al., 2008) (Hu...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...

...small (12-15 kDa) single-domain proteins that can bind effectively to protein targets, similar to how antibodies... sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky...candidate sybodies in their coat protein to screen for binding to the target protein. The protocol describes going...identified nanobodies that bind to coronavirus spike proteins (Wrapp et al., 2020), which sit on the surface...host cells. To do this, they injected the spike protein from two different coronaviruses into a llama over...special camelid antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  ...antibodies against unstable targets like membrane proteins that may unfold due to camelid high body temperature...

.... Spike protein, variants, and pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ...spike protein was developed based on 100 structure-guided spike designs and has improved protein yields...and identified interactions of these proteins with human proteins. Their deposited plasmids expressed ... more about mapping the SARS-CoV-2 - human protein-protein interaction Find tips for remote collaborations...over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman ...scientists have developed ways to pseudotype the spike protein onto another virus. With the pseudovirus, scientists...pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited the plasmids used for this study...

...processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such generic processes...References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using efficiently-delivered...Hiroki, Sebastien Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for Targeted...resistance, mediated by a single base pair change in DNA gyrase, CRISPR phagemids were cytotoxic only for resistant...

...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...fluorescent proteins, antibiotic resistance, and luciferase. References Gaudelli NM, Komor AC, Rees HA,...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...

... and a protein standard. The density of the antibody bands can be measured against the protein standard...predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411-23. doi: 10.1002/... A. Pathak, Christopher J. Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization...applications you are planning, cost, and the amount of protein you need to produce.  The most common yeast strain...pastoris) because it secretes fewer of its own proteins, making the purification process simpler. Due ...incubation time to maintain cell viability and increase protein production (Schwarz et. al., 2020). Harvest and...quantification. Many spectrophotometers have an IgG protein setting. If yours does not, be sure to set the ...

...detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield, 2014). Traditionally,...chain for the antibody and when translated into protein will assemble into a fully functional antibody....encoded in a plasmid. (Right) An assembled antibody protein binds an antigen. Image from Fvasconcellos. ...genes for antibody expression. If starting from a protein sample (for instance, affinity purified antibodies...well in animals because of similarities to host proteins. After the antibody of interest has been cloned..., Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, Nguyen K, van der List D, Murray KD, Engebrecht...

...there are many protein-based toxin-antitoxin systems. One of the most well known protein-based toxin-antitoxin...specific sequences in RNA resulting in a blockage of protein synthesis. The antitoxin MazE, which binds and ...Toxin-antitoxin systems are not just restricted to the protein realm. Let’s take a look at a toxin-antitoxin system...additions to constructs that are used to drive high protein expression in vivo. Expression plasmids are often...PMC39188. Kamada, Katsuhiko, Fumio Hanaoka, and Stephen K. Burley. "Crystal structure of the MazE/MazF complex...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or... replication by binding the GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results...

...cells in a population. It can be used to measure protein expression, identify rare cells, or even sort out...Alyamani, A., Kalamegam, G., Ahmed, F., Abbas, M., Sait, K., Anfinan, N., Al-Wasiyah,  mohammad khalid, Huwait...i1.23 Jhunjhunwala, S., Aresta-Dasilva, S., Tang, K., Alvarez, D., Webber, M., Tang, B., Lavin, D., Veiseh... J. A., Campillo, J., Moya-Quiles, M., Minguela Puras, A., Pons, J. A., & Muro, M. (2018). In vitro intracellular...