...interaction between a bisarsenical dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine...systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with... as intact tissue. Some chemical labeling strategies such as the bisarsenical dye (Figure 1a) and FAP ...Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical biology 3.3 (2008): 142-155. PubMed... at the Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry...fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer...synthetic fluorophores. Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing...

...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect...lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox lentiviral vectors:... to reprogram. Basically, there’s no universal method that can handle all applications of iPSCs, but there...generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem cell reprogramming... to verify there’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium ...creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing...unavoidable heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene...

... (enChIP) using CRISPR." Biochemical and biophysical research communications 439.1 (2013): 132-136. PubMed...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...26848818. Fujita, Toshitsugu, et al. "Identification of physical interactions between genomic regions ...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...

...transfected (see Technical Tips). Minigenome replication and transcription In order to replicate the minigenome...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...5’ ends of the genome which are required for replication and transcription (Figure 1). All viral genes...cis-acting signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase... in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...

...resistance-mediating polymorphisms in African samples of Plasmodium falciparum." Journal of clinical microbiology 51.8 (...furiosus." Acta Crystallographica Section F: Structural Biology and Crystallization Communications 61.12 (2005... In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols,...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...rounds of melting and annealing allows linear amplification of the probe ligation product. With the right...

...D) teams at pharma companies carry out preclinical and clinical research to discover and test the best...years. Complicating these challenges is the fact that they occur in the context of significant clinical...training in reviewing literature, and critical analyses skills were applicable to my cases. I can directly use...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...efficient ways of delivering healthcare. Clients typically engage consultants as short-term experts to work...acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness among...

... receptors which play a critical role in development and are often implicated in disease progression. ...inability to replicate the spatio-temporal precision with which signaling events occur in a physiological setting...brighter mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to engineer...observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly induction heterodimerization...flexible cloning process provides improvements to classical cloning methods, particularly for complex transgenesis...enabled the group to perform independent two-color optical excitation of neurons. The paper describes five...setting. Genetic techniques typically rely on overexpression or knock-down, where signaling dynamics are...

...rather than being geographically constrained to the brain. In cases where it’s critical to minimize or eliminate...recommended for all systemic capsid applications. For example, AAV-PHP.eB is typically administered at a total dose...your specific application and animal model.  Other model organisms Tropism and efficacy can vary depending... 2023 * indicates viral preps available. Mechanisms of crossing the BBB are indicated in parentheses...crossing into the brain. While helpful from a biological standpoint, it makes delivering AAVs to the brain...throughout the organism to target large or diffuse biological structures. Of primary interest here are systemic...suitability of a systemic capsid for an individual application is dependent on a number of factors, including...

...FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have remained...Identifying new ligands for orphan GPCRs is of significant medical interest, however efforts to do so have been...GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones for lentivirus...mutant library generation in several biotechnologically and clinically relevant bacterial species. pORTMAGE...backgrounds can lead to disparate physiological effects and complicate any metabolic study. The Ralser ...brief articles highlight the main features and applications of a partiular plasmid or set of plasmids. We...Wu Lab library can be found in the associated publication. Ma, et al. Cell Rep. 2015. PubMed PMID: 26190106...

...protein site-specifically to alter its function, mimic a biologically relevant modification, or add in ...function relationships or, in more practical applications, allow a protein to be more effectively used in ...presents a significant barrier for those wishing to use it as a tool. In short, while the technical skills...proteins, you’re probably quite interested in the canonical 20 amino acids. But in your quest to learn more...code expansion (GCE): techniques in which non-canonical amino acids (ncAAs, also sometimes called unnatural...in fluorescent or chemically reactive side chains, all of which can help determine structure and function...was developed into an E. coli system, with the noncanonical amino acid in the media and the tRNAs and synthetase...

.... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several factors contribute...probe, it is the first choice when selecting a genetically encoded fluorescent tag. There are, however, ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...of these strategies works best for a specific biological system under anaerobic conditions needs to be...adding GFP may alter diffusion rates. Smaller genetically encoded probes are based on cofactor binding ...tags are peptides that are engineered to bind specifically and with high affinity to fluorophores. Examples...

...or feedback. One of the most significant shifts in scientific communication is the rising use of preprints...arXiv.org), but have been only adopted by biological and biomedical scientists (see: bioRxiv.org or medRxiv.org...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, Twitter...publishing in a journal.  Accelerating Scientific Communication Preprints have been common in fields like math...increasingly growing time from submission to publication. It takes many months, often more than a year...

...people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...background, your issue is likely technical. White circles, for example, could indicate that you had air trapped...Sensitivity in Western Blotting. Journal of Pharmacological and Toxicological Methods, 64, 233–237. https://doi....to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself... be done at 4 °C or on ice, whichever is more practical. If your lysing method is not resulting in your...nuclear- or DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins ...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...

...Diecke, S., et al., Recent technological updates and clinical applications of induced pluripotent stem...opened up vast possibilities for the clinical and therapeutic applications of iPSCs. The generation of human... patients, specifically children and patients with skin disorders for research and clinical trials, as...derivation of clinical grade hiPSCs, reprogramming methods that do not involve modification of the original... OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. ...system thus provides a practical way to generate transgene-free, virus-free clinical grade hiPSCs from easily...therapy trials and remains a safety concern in the clinical field [4]. Three major reprogramming methods are...

...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...library can drastically impact what conditions it needs to properly amplify.  Amplification protocols therefore... prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our...process for using pooled lentiviral libraries. A) Amplification, validation, and lentiviral generation of a ...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...

...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...your grant application to your peers and collaborators can also help you get some critical insight into...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...someone else in the department) to read over your application and give you feedback. They’ve most likely been...

...experiments with too few replicates leading to suspect statistical significance (you can read more about...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...formulating new biological concepts. However, rates of reproducibility for preclinical studies, as reported...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...more practical) is pre-registration and review of experimental design as part of the publication process...on solutions to reproducibility issues in the biological sciences and featured speakers from academia,...Overview - Jeffrey S. Flier, Researcher at Harvard Medical School, former dean of the faculty of Medicine ...

...production and purification that yield high titers (see also AAV protocols for production, purification and titration...higher specificity and reduced technical variability compared to chemical tracers. rAAV2-retro also has...ability to spread trans-synaptically on to subsequent neurons. AAV vectors are replication incompetent and cannot...intracerebral application, which is more invasive and has a much higher risk of complications. References...between different brain regions. For example, communication between the hippocampus and the prefrontal cortex...memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional...intracellularly spread away from the site of application and aid in visualization of the morphology of...

...research? I grew up knowing little to no biological or biomedical scientists and had no idea what the daily... global problems. Biomedical research, for instance, contributes heavily to medical practice, and understanding...comes your way. Think critically about whether it’s something you want to dedicate your time and energy...These conferences have a lot of programming specifically dedicated for career advancement, and for students...entire page dedicated to different career options from science policy to science communication and much ...the Daily Psychological Experiences of First-Generation College Students. Social Psychological and Personality...hazardous liquids. Working in a lab is nothing like a typical part-time or 9-to-5 job. I had to ask to find out...

...get there and more practically, when do you need to be there? A typical biomedical postdoc fellowship ... findings that fail to replicate. Plan experiments logically and strategically to increase your rate of...theJanelia Research Campus of the Howard Hughes Medical Institute. Today's postdoctoral fellows (PFs) face...need? Multiple first or corresponding author publications (published, not in preparation) A history of...submitted manuscript in a high-profile journal typically takes 6-12 months depending on the revisions and... to be ready to write by Year 3.5. Thus, it is critical to get an experimental system up and running very...The other items will follow. If you get more publications, you will be more competitive for fellowships...