...diabodies to be used for assembling protein nanostructures. Diabodies readily co-crystalize, a major advantage...only affinity reagent on this list derived from nature. Single-domain antibodies are extremely small at...but can have weak signals due to their monovalent nature. They are stable up to 80°C and can refold correctly...Books. Bates A, Power CA. David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody ...single-domain antibodies. Annu Rev Biochem. doi: 10.1146/annurev-biochem-063011-092449.             ...

...and designing future experiments. Being able to work through these types of bigger picture problems systematically...problem solving!  You might think that being able to figure out what went wrong with a PCR is a pretty niche...helping to resolve problems, but even so, I wasn’t sure how to best define this skillset. After sifting ...your problem solving skills can help convince a future employer that you are well equipped to work through...

... to increase capture. Longer histidine tags are also recommended if you need a very pure sample. Because..., alternative fusion tags were used, such as S. aureus’ Protein A (Moks 1987). These tags, however, tended...denaturant. Not all proteins will refold once denatured, however, so take care when using this method....purification of human insulin-like growth factor I from culture medium of E.coli. Bio/Technology. 1987, 5:379-382...

...mammalian cells. Here, we will cover how these structures are generated, why it’s important for cells to...proteins of interest. RNAi has even made it into agriculture as species-specific pesticides and insecticides...one strand of a dsDNA molecule; the 3-stranded structure produced is known as an R-loop. During transcription... transcription-coupled R-loops are transient in nature and will dissociate when gene transcription is ...vulnerable to various forms of DNA damage, and these structures can in turn lead to genome instability. Thus,...

...career in science. Supervisors in the academic infrastructure are not always as effective in mentoring for.... Talk to the coach about their “style” and make sure this is a style that will help you. A good coach...also a very popular speaker and workshop leader. Lauren Celano of Propel Careers is not a professional ...afraid to ask and to make an investment in your future. Additional Resources on the Addgene Blog 6 Steps...Little about The Academic Labor Environment from the Future of Research Symposium Resources on the Addgene...

...expression of certain viral components; according the manufacturer these changes result in yields up to 25x higher...harvest is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios...While this option is attractive because the same procedure for a single 10 cm2 dish can be easily applied...consequently, this may not be the best method when pure virus is required downstream. When assessing lentiviral...titering your lentivirus Visit the viral vector featured topic page Resources on Addgene.org Lentiviral...

...challenging for amateur scientists. Planarians, wormy creatures commonly found in freshwater ponds, provide a ...preparation of the tissue sample, several washes, and temperature changes to visualize the expression of specific... purified dsRNA with liver paste, and feed the mixture to the worms. The phenotypes of RNAi knockdown ...use these published protocols and want to share pictures, feedback etc. please write to [email protected]... double-stranded RNA in Caenorhabditis elegans. Nature, 391 (1998), pp. 806-811. PubMed PMID: 9486653....

... very little signal detected along the neurite (Figure 1.) Moreover, the group was able to demonstrate...are available at Addgene. Shemesh OA, et. al. Nature Neuroscience. 2017. PubMed PMID: 29184208.        ...methylxanthine drugs like theophylline. To facilitate future work with this goal, the Barrick lab has deposited...fLuctuation INcrease by Contact (FLINC)-based biosensors measure fluctuations of TagRFP-T fluorescence intensity...use to detect PKA activity can be generalized to measure phosphatase activity, second messenger activity...

...optimize more protocols in the future - something that will be key for our future growth. One of the biggest...pipette samples in and out of it, you can set up a procedure for it using the Biomek FxP. For example, we added...us to put a label on each tube by hand. We’ll be sure to tell you about more lab updates as we develop...have any great lab automation tips and tricks, be sure to share them in the comments section.   Additional...

...particles at the cell membrane. Mature Nanoblades then bud into the cell culture supernatant and can be purified...following four components into 293T cells. Figure 1: The key components of Nanoblades. Image from...iPSCs and BM cells after Nanoblade treatment as measured by their maintenance of pluripotency marker expression...Nanoblades loaded with Cas9-sgRNA ribonucleoproteins." Nature communications 10.1 (2019): 45. PubMed PMID: 30604748...

...each part of a split fluorescent protein (FP) and measure the signal produced when the candidate proteins...interaction brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular...Addgene is here to help!  General Design Figure 1: Two proteins are fused to the FP(11) and FP(...interact, the FP fragments can assemble the full structure of the fluorescent protein, producing fluorescent...number of strands in the canonical beta-barrel structure. Most frequently, the proteins are split between...

...modifications). Ready to get started?   Figure 1: Break free from the ball and chain!   Epigenetics... lot of things were cool: Walkmans, Back to the Future, and big hair. To what was perhaps a slightly smaller... (Hrit et al. 2018, Kaas et al. 2013). Figure 2: CRISPR TET-based epigenetic editing. In reality...expression (Sapozhnikov and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks DNMT1 methylation...some human cancers from their normal counterparts. Nature 301, 89–92. https://doi.org/10.1038/301089a0. Galonska...

...format that was stable for up to a year at room temperature (Pardee et al, 2014). As a demonstration of the...switches and amplification primers. This helped make sure that we had a number of successful sensors to draw...switch-based RNA sensors within an extensive infrastructure of molecular and electronic companion technologies...without purification or concentration of the virus. Future work Going forward, our short-term efforts are ...rapid prototyping and sensor assembly pipeline for future emerging pathogens or health crises. Many thanks...

...DH5α, grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of your virus, perform a diagnostic...ultracentrifugation, you can reduce the volume of cell culture medium on the packaging cells immediately following...5 mL medium on a 10 cm plate). By reducing the culture volume into which the virus is released, a more...experiments later. Stay tuned to the Addgene blog and be sure to check out our viral vector guide pages to get...DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology." Journal of clinical...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair...Mismatch positions are indicated below each graph. Figure adapted from Kleinstiver et al., 2016. Until... failed to induce off-target editing in a cell culture model. What we still don't know about Cpf1 In ... its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively...specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology (2016). PubMed PMID: 27347757. Find...

...industry through epigenetic effects on the Cover of Nature. So what is epigenetics and what tools are available... contain the same DNA but have very different structure and function, and can have a specialized role ...syndrome is characterized by low muscle tone, short stature, cognitive disabilities, problem behaviors, and...specifically to the histone protein of interest (in the figure above, H3 at K9 or K27). Joined to this by a flexible...underlies the mantled somaclonal variant of oil palm." Nature 525.7570 (2015): 533-537. PubMed PMID: 26352475...

... you can afford them). I am talking about making sure the audience is interested, educated and talking...interesting questions and topics in advance is key. Make sure all your panelists have the correct date, location...colleagues to events for this purpose. If possible, make sure the panelists are prepared to stay around to meet...the moderator and program organizers can help make sure the event stays on schedule. People can only sit...the attention of the audience – phones come out, postures shift, they are checking out. If you can, plant...

...Snapshot: Negotiations When Cultures Collide: Managing Successfully Across Cultures by Richard D. Lewis ... partners and how can Addgene work with them to ensure we create a win-win situation? Learning about and...Whether you are serving on academic committees as a tenured professor or signing contracts when working for...Salary negotiations will be covered separately in a future post.   Resources Maria recommends: Getting...

...pitted against CRISPR head-to-head.  Exciting features of NgAgo First, unlike Cas9, NgAgo does not require...incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes ...for the trickiness of NgAgo is the inability of mature NgAgo protein to load an ssDNA guide. It is possible...form a complex with a 5’-P-ssDNA guide at room temperature. Until that time, those who have been lucky with...using the Natronobacterium gregoryi Argonaute." Nature biotechnology (2016). PubMed PMID: 27136078. 2....

...from a unique promoter, multicistronic plasmids ensure that any cells that are positive for your marker...translation from an internal region of the mRNA. In the figure above, you can see that the IRES element acts as...you to spend less time researching plasmid basic features and spend more time developing the clever experiments...sequence derived from poliovirus RNA. Pelletier et al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment... mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity...

...look at structure, impediments, the representation of women on committees, and how the culture of the ... promoters and terminators, but then they aren’t sure how to convert their own coding sequence to the ...science based around those technologies in the near future. Science on Twitter Addgene: You are active on ...Twitter account recently inspired by an academic in literature who gave a talk to the women’s research network...

...proteins associated with the IRF-1 locus. Figure 1: Schematic of enCHIP. Image from Fujita and Fuji...affinity purification in situ of regulatory elements (CAPTURE) (Liu et al., 2017). This system utilizes dCas9...through proteomics and the chromosome conformation capture (3C) technique. In addition to 3xFLAG and biotin...FSHD), a disease for which there is currently no cure (Campbell et al., 2018). Find plasmids from the ...Central PMCID: PMC6286148. Liu, Xin, et al. "In situ capture of chromatin interactions by biotinylated dCas9...

...an antibiotic resistance gene is a mechanism to culture only bacteria which have retained a functional ...formation on agar plates, less stable at high temperatures and in acidic environments.   Carbenicillin   ...can be successfully used for plant and bacterial culture, but some types of bacteria, such as SHuffle cells...all-in-one antibiotic — it is usable in eukaryotic cell culture, yeast, bacteria, and even plants! The zeocin resistance...., & Brooks, G. F. (1986). Effect of storage temperature and pH on the stability of antimicrobial agents...

...you don’t have plasmid ID numbers, or you aren’t sure if any plasmids from a paper have been deposited...plasmid search and found the results confusing, I’m sure you’ll find Addgene’s newly launched search engine...marker used to grow plasmid containing bacterial cultures Bacterial Resistance: Use this to choose the antibiotic...Editing brings us to a page with a basic overview and figure, followed by a convenient table of plasmids specifically...resources on the Addgene blog Learn more about other features on Addgene's website ...

...applications. Read on to learn more about the current (and future) applications of lentiviral vectors! Lentiviral...popular for targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system ...to create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important... Dull et al. created the 3rd generation system (Figure 3), which differs from the 2nd generation in two...Gluckman E, Bushman F, Hacein-Bey-Abina S, Leboulch P. Nature. 2010 Sep 16;467(7313):318-22. PubMed PMID: 20844535...

...are required for replication and transcription (Figure 1). All viral genes are removed and replaced by...transcription, as it would for a full-length viral genome (Figure 2). While it is technically possible to encode ... luciferase. Firefly luciferase can readily be measured to quantify (indirectly) transcription performed...result in significantly reduced activity (4). To ensure that you end up with appropriate plasmid ratios... not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional...

... grant readiness, identify grant opportunities, secure grant awards, and manage grant projects and funds...management, we track grant opportunity deadlines, and we ensure that grant reports are submitted and project evaluations...field of grants. It is no surprise that the U.S. Bureau of Labor and Statistics shows that employment of...program didn’t stay in one place within the city infrastructure. My career path changed when I was asked if...She and her team at J. Miller & Associates have secured more than $70 million dollars in grant funding....

...plasmids of these systems to Addgene in the near future. Addgene: Has your lab been working on any other...development in biology and hope to develop more in the future. Involvement in the Scientific Community Addgene...papers submitted to journals. In addition, it is my pleasure to interact with my colleagues in meetings of ...institutes in different places have very different cultures. In Basel, I felt that the time flows much slower.... I hope I can help change the situation in the future. Addgene: What advice do you give to post-docs ...

...can direct people to the AMA in the future and link to it in future blog posts on these topics. Others ... new questions and topics to write about in the future. The AMA was also cataloged for permanent reference.... I have a paid account which gives me all the features I need to save, organize and easily find the resources...on the internet or receive via email. The cool features are too many to describe here, but I have over...

...candidates have seen an actual chalk talk. Their first exposure to a chalk talk is usually their own. This is ...in the remaining time. The typical chalk talk structure is: What is the big question or mystery that ...livers for transplants. Growth of new livers in culture could resolve this shortage." What is the knowledge...Perfect your story and delivery. Design simple figures and diagrams to illustrate your vision. Practice...beyond the whiteboard. You may wish to create a sculpture or print a 3D model to better illustrate your ...