...limited biological materials. Problems with Chip-seq ChIP-seq typically requires a large amount of input...enough material for a ChIP-seq experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking...immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where a protein...in many model organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it...patterns that overcomes some of the limitations of ChIP-seq. CUT&RUN, which stands for ‘Cleavage Under Targets... using the Drosophila wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings. Collecting...All in all, CUT&RUN offers a great alternative to ChIP-seq, especially in situations when it is technically...

...secondary antibody. Chicken IgY This RANbody reporter consists of two chicken IgY domains. Chicken IgY domains...antibodies. Chicken IgY RANbodies are easily detected with a labeled anti-chicken antibody. Chicken IgY RANbody..., H2A2B Myc Tagged RFP, H2A2B Chicken IgY (Y) Labeled Anti-Chicken IgY antibody Staining cultured cells...three varieties: HRP, Spaghetti Monster, and the Chicken IgY domain, which are discussed in more detail ...the Key Features of HRP, Spaghetti Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection... anti-rodent or -rabbit Detection with an anti-Chicken IgY antibody is approximately as sensitive as detecting...

...FP-protein fusions (FP-chimeras) is that they undergo normal protein turnover. FP-chimeras are continuously ...degraded within the cell, so at any given time, an FP-chimeric protein may be at any one of many stages of synthesis...turnover or temporal expression using standard FP-chimeric proteins. Photoactivatable fluorescent proteins...bright state to a dark non-fluorescent state (“quenching”). The photoswitch can occur multiple times by...reversible PA-FPs also display this “negative” photoswitching. In 2008, scientists used mutagenesis screens...V157G / M159Y / F190S) (7). Padron’s “positive” switching behavior is the opposite of Dronpa; when exposed...monomeric PA-FPs, there is less chance that the chimeric protein will oligomerize, disrupting cellular ...

...and most machines have a protocol that automatically performs the normalization step.  Machines Most general-use...different brand of qPCR machine, especially if you’re using a rapid-cycle machine such as a LightCycler,... LUX fluorogenic primers Low Single self-quenching fluorescent molecule connected to a primer  ...previously-generated validation data, be aware that different machines, samples, and treatments may affect the assay....running a multiplex reaction or on a rapid cycle machine. Melt Curves An advantage of Sybr chemistry is ... by bubbles in wells, variations in volume, or machine surge errors and allow for well-to-well normalization... likely need to re-optimize protocols for that machine. (Teo, et al, 2002; see paper for more information...

...Streamlined VchINT expression constructs. (B) High integration efficiencies achieved with VchINT, at two ...multiple distinct targets could be achieved (Vo et al., 2020). VchINT was also shown to be active in various...Vibrio cholerae (V. cholerae INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The ...Type I CRISPR-Cas systems, however, Cascade from VchINT does not recruit other nucleases (such as Cas3)...; although 5’-CC-3’ is one of the best PAMs for VchINT, many other PAMs are also efficiently recognized...engineering In the Sternberg group’s initial report of VchINT, three separate plasmids and multiple promoters... al., 2020. In E. coli, the new streamlined VchINT system was shown to catalyze insertion of mini-...

...origin of replication and the replication control machinery (Need a refresher on the Origin of Replication...they compete for the same replication control machinery within the cell. Most plasmids encode a negative...through inhibiting translation of replication machinery proteins, or directly inhibit replication by binding...origin of replication and blocking replication machinery (del Solar, 1998). As the copy number of the plasmid... structure that physically blocks replication machinery when plasmid copy number is high (del Solar et...replication of each plasmid using unique replication machinery. However, when plasmids have the same origin of...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like...

...Vierock et al., 2021). Step-function opsins are chimeric ion channels that turn on with a light-pulse at...with greater spatial and temporal control than is achieved by traditional pharmacologic methods. A number...Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS (OptoXRs) (Tichy et al., 2019), photoswitchable...remotely-tethered ligands (PORTLs). OptoXRs are chimeras of the extracellular domain of endogenous light-sensitive...signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes a conformational...secondary signaling pathways with light. This is achieved by coupling endogenous signaling molecules with...Protein-Coupled Receptor Signaling. In D. Niopek (Ed.), Photoswitching Proteins (Vol. 2173, pp. 21–51). Springer US...

...previously developed small molecule degraders that achieve selective degradation of endogenous proteins (notably...Hippel-Lindau (VHL) to bring the endogenous degradation machinery into close proximity with the target protein, ...to co-opt the endogenous ubiquitin proteasome machinery to degrade any protein. We are particularly excited...FKBP12F36V-fusion and the ubiquitin proteasome machinery through binding with cereblon, leading to rapid...of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific knock-in of FKBP12F36V. Detailed...your lab Confirm the functionality of your fusion chimeras For each protein-of-interest, it is important ... evaluate degradation of the FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated...

...Technique What It Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome...Aneesh's research interests include proteomics, machine learning, and visualization for big biology. GPS...detect modified histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and...formula to a real-world experiment. We’ll start with ChIP-seq data from the ENCODE project, find enhancers...enhancers. A real-world experiment Suppose you run a ChIP-seq experiment (think “GPS for proteins”) for NANOG...transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant binding peaks... (loops) and tertiary structures (globules) to achieve exquisite compaction—on the order of 700 terabytes...

...conditions. Common issues include photobleaching, non-specific binding, and quenching, which can be mitigated through...photostability, and most importantly, a photoswitching ability. This photoswitching refers to the ability to ‘toggle... must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility due...peak wavelength can also decrease the rate of photobleaching. Interaction with receptors: Understanding ... technical manuals. Specific applications and matching dye/ligand choices For effective live-cell imaging.../doi.org/10.1007/s41048-017-0037-8 Lucidi, M., Capecchi, G., Visaggio, D., Gasperi, T., Parisi, M., Cincotti..., M., & Ghods, R. (n.d.). Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568...

...microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple but limited...when FRET occurs. By photobleaching the acceptor, you release the donor's quenching and the fluorescence...An EAMNET Teaching Module http://www.embl.de/eamnet/downloads/modules/FRET_teaching_module.pdf Day RN, ...brightness of a fluorophore pair, donor:acceptor stoichiometry, and cross-talk between the two fluorophore ...has five-fold less brightness than YFP. The stoichiometry of donor:acceptor is difficult to address especially...commonly used to measure FRET: the acceptor photobleaching method and the fluorescence lifetime imaging...

... was weird like that,” says Caitlin. A love of teaching also runs through Caitlin’s blood. “I come from...faculty members, I helped grad students who were teaching for the first time. I walk[ed them] through their...them advice, [and helped] proctor exams.” These teaching experiences led Caitlin back to being a student... signed up for a 10 week class at Iowa called “Teaching Your Research.” This course has participants take...ground up.”  Science writing and editing These teaching experiences definitely weighed in Caitlin’s post-grad...but in the end, writing won out. “I considered teaching at a liberal arts school but I’ve always enjoyed...off: “I spent about 6 months just editing and searching for a job and I stumbled upon eScience by luck...

...it’s true that adding histones and chromatin architecture to every diagram portraying some aspect of eukaryotic...when I recently attended the 2018 Chromosome Architecture and Chromosome Organization Keystone Symposium...but also specifically study it. They might be researching enhancer elements that control the transcription...posters I saw at the 2018 Keystone Chromosome Architecture and Chromosome Organization. These aren’t the...’re looking for on this list, please consider searching directly on our website. In-exhaustive Chromatin-related... that promotes homologous recombination. Gong F, Chiu LY, Cox B, Aymard F, Clouaire T, Leung JW, Cammarata...Crabtree GR Cell. 1998 Nov 25. 95(5):625-36. An architectural role for a nuclear noncoding RNA: NEAT1 RNA ...

...down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues showed that a strong protein denaturant...denaturant, guanidinium thiocyanate, did just this (Chirgwin et al., 1979). They developed a protocol meant...ultracentrifugation altogether. Chomczynski and Sacchi showed that RNA could be effectively separated ...method are still widely used today (Chomcynski and Sacchi, 2006).  View our protocol for RNA extraction! ...ammonium sulfate on the activity of ribonuclease A. Biochimica et Biophysica Acta (BBA) - Enzymology 341:484.... https://doi.org/10.1016/0005-2744(74)90240-x Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979...https://doi.org/10.1021/bi00591a005 Chomczynski P, Sacchi N (1987) Single-Step Method of RNA Isolation by...

...Pubmed PMID 23887178. 7. Watabe-Uchida M1, Zhu L, Ogawa SK, Vamanrao A, Uchida N. Whole-brain mapping of direct... of us learned in biology is that the body’s architecture and plumbing are important. We started with ...oxygenates the body. However, in neuroscience, this architecture is frequently still an open question. The connections...way, but rather just labels the whole brain. To achieve labeling of only first-order connections to the...cell type specificity of the starter cell can be achieved by using EnvA-pseudotyped RABV, and only expressing...supplied in trans. Monosynaptic RABV spread can be achieved by modifying the wild-type RABV genome, which ...populations in two specific brain regions (Watabe-Uchida et al., 2012). They could also quantify the strength...

...fluorophores to block out of focus fluorescence from reaching a detector or restricting fluorescence to specific...fluorescent light passes through a pinhole before reaching the detector. The pinhole is specifically placed... the focal volume. This leads to more rapid photobleaching and a reduction in signal intensity. 2-Photon...Photon microscopes restrict excitation (and bleaching) to a single focal point. This is accomplished by ...the sample is excited at one time, limiting photobleaching. Similar to widefield, the entire field of ...coverslip (4). Essentially, an optical section is achieved that boosts contrast by filtering out emission...photons, a higher signal to noise ratio can be achieved (6). Super-resolution techniques such as Structured...

...formerly known as Pichia pastoris). If your goal is protein isolation, then the MoClo Pichia Kit will get ...discuss. If you can think it, MoClo might help you achieve it! MoClo applications and specialized tools Synthetic...a broad range of parts, including copy number machinery, chromosomal integration modules, and many more...to study the effects of protein degradation on chimeric antigen receptor signaling (Kim et al., 2024)....can dream up an experiment, MoClo can help you achieve it! More recently, a CRISPR MoClo kit termed Fragmid...fungi, bacteria, protists, plants, and animals (Chiasson et al., 2019). Many plasmid elements such as promoters...Ulrike, et al. “A Modular Toolkit for Generating Pichia Pastoris Secretion Libraries.” ACS Synthetic Biology...

...spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP). In IP/MS, a target protein is first immunoprecipitated...identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by... patterns of the target protein. Both IP/MS and ChIP-seq methods provide a wealth of information about...all-purpose “tag antibody” can be used for downstream IP/ChIP assays. Creating epitope-tagged clonal cell lines...Using our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency across different ...clonal knock-in lines that we now use for IP and ChIP assays. Tag 96 genes at a time using Custom Synthetic...transfection/delivery step needs to be optimized to achieve high knock-in efficiency. To calculate the tagging...

...molecule-mediated chromatin immunoprecipitation (enChIP) because any engineered DNA-binding molecule, such... the IRF-1 locus. Figure 1: Schematic of enCHIP. Image from Fujita and Fuji, 2013. Prior to...molecule (Hoshino and Fujii, 2009). Other forms of enChIP, that do not use dCas9, require Zinc-finger or ...purification of a specific genomic region, other forms of enChIP, and additional research conducted in the Hodaka...molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR." Biochemical and biophysical research...molecule-mediated chromatin immunoprecipitation (enChIP)." Scientific reports 3 (2013): 3171. PubMed PMID...associated with telomeres using a combination of enChIP and RNA sequencing." PLoS One 10.4 (2015): e0123387...

...devastating afflictions. Elia Di Schiavi - Spinal muscular atrophy Dr. Elia Di Schiavi is a Research Scientist ...mechanisms behind the disease are unclear. Dr. Di Schiavi plans on using C. elegans as a model to identify...disruption. As part of his work in C. elegans, Dr. Di Schiavi will use Addgene plasmids to characterize smn1 ...behind SMN1 and its interaction partners, Dr. Di Schiavi will be able to better choose which genes and pathways...SMN1 and its partners in mouse cell lines. Dr. Di Schiavi hopes that these studies will accelerate the development...Website.  We are proud to provide support to Drs Di Schiavi and Nabavi and will continue to work to help those...