...Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins such ...anti-FP antibodies. GFP emits light in only a single channel. In multi-color imaging experiments, the second...making them difficult to use in a multicolor/multichannel imaging set-up. In contrast, smFP tags can be...University Press, New York. pp 719. 3.Rizzo, Mark A., Michael W. Davidson, and David W. Piston. "Fluorescent ...4. Shaner, Nathan C., George H. Patterson, and Michael W. Davidson. "Advances in fluorescent protein technology...

...gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb...the group to focus their efforts on SaCas9 characterization for in vivo studies. One of the pitfalls of...genes, they saw indel formation and phenotypic changes as early as 1 week post-injection. Livers from ...jejuni (CjCas9) is the smallest Cas9 ortholog characterized to date. Kim et al. successfully used CjCas9...transgenes might seem daunting, it’s actually a challenge that the field has overcome with split AAVs. In...

...in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily in...into genetic compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). ...fluorescently labeled, allowing researchers to observe changes to these tissues under different experimental conditions...time and space, and was also used to precisely characterize developmental defects in the one-eyed pinhead...2016. 219-244. PubMed PMID: 27443928. Harland, Richard M. "A new view of embryo development and regeneration...

...share all of these new organisms continues to be challenging. Organism specific viral vector systems would...thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community...So, think beyond Drosophila melanogaster or Saccharomyces cerevisiae (both fine research organisms) -  ...social bonds that they form including genetic mechanisms that control who a female wants to mate with ... first? They are starting by seeing if they can change long legged lizards into short legged lizards.   ...

...to interfere with the function of resident ER chaperones and may impact cellular function or possibly ...For example, the formation of inappropriate inter-chain disulfide bonds between FPs blocks Golgi complex-localized...protein activity. We encourage investigators to characterize FP-fusion proteins functionally relative to ...moxFPs. The inherent brightness and spectral characteristics of the moxFPs are comparable to the non-optimized...postdoctoral fellow at the University of North Carolina at Chapel Hill. Under the co-mentorship Blossom Damania and...

...sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...better response kinetics and photochemical characteristics than previous NIR calcium reporters iGECI and...   Matlashov ME, et al. Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium... B) LiLac responses are highly resistant to pH changes. Image adapted from Koveal et al. 2022, under CC-BY...

...found it easy to drop into a chair and chat with me remotely via an audio chat (Fig. 4).  Learn: This works...small operation, so sometimes it can be a big challenge. But ask any filmmaker or videographer and they...you can usually see a director sitting in their chair not watching their actors live on set, but peering...

...centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators can...: 2388-2393. Huang, Shijie, Xuechen Zhu, and Charles E. Melançon III. "Detection and Quantification...metalloproteinase: López-Pelegrín, Mar, et al. "A novel mechanism of latency in matrix metalloproteinases." Journal...size from 4 to 156 kDa, enabling the user to characterise the efficiency of their cell-free system in ...5373-5383. Walkiewicz, Katarzyna, et al. "Small changes in enzyme function can lead to surprisingly large...

...designed to be a helpful desktop reference. Each chapter brings together all of our 101 blog posts on an...to antibodies as research tools, you may find “Chapter 1: What is an Antibody” quite useful. If you’re... design your first flow cytometry experiment, “Chapter 7: Sorting” is there to help guide you through ...find an antibody that works, we highly recommend “Chapter 3: Finding the Right Antibody for Your Experiment...

...positively charged groups, such as aldehydes in fixed samples. To address this, block positively charged groups...the label control for all new samples, protocol changes, or when high background labeling occurs (Burry...critical for data interpretation, especially when changes in protein expression are the experimental readout...       References and resources References Burry, Richard W. “Controls for Immunocytochemistry.” Journal ...

...promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes and promoters... different receptors, co-receptors, and other mechanisms to gain entry into the cell (Dudek et al., 2018... different brain regions.  Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons...the region you want to cover - and as your titer changes, so may your needed volume (de Backer et al., 2010....2014.00076 References and Resources References Aschauer, D. F., Kreuz, S., & Rumpel, S. (2013). Analysis...

...added, it binds tTA, resulting in a confirmation change that prevents binding to the promoter and turning...added, it binds cTA, resulting in a confirmation change that prevents binding to the promoter and turning...this way, you can interrogate the activity of uncharacterized promoters. Similarly, placing UAS upstream ...1472-6750-6-43Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Cumate-inducible gene expression system for ...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the...First, you’d construct a plasmid with your desired changes, long homology arms and a selection marker, then...template. Although effective, ivTRT may be more challenging for sequences with complex secondary structure...-purified DNA template.   Many thanks to Channabasavaiah B. Gurumurthy, Masato Ohtsuka, and Rolen M....PMC5434640 Miura, Hiromi, Rolen M. Quadros, Channabasavaiah B Gurumurthy, and Masato Ohtsuka. “Easi-CRISPR...

...had record readership with over 75,000 views. Changing Labor Laws Bring Increased Postdoc Wages Our most...information for anyone considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,” Future...for some of these institutions to manage these changes quickly and harmlessly. Learn much more about the...Reasons to Use Reddit for Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope...

...to face global challenges, we hope to empower those that need to face these challenges locally. We look...themselves on the front lines of emerging global challenges. However, these same researchers face significant...international distribution As you can see from the chart below, we are making great progress toward spreading...

...Antibodies are composed of two light chains and two heavy chains. The ends of the branches contain the...variable, allowing exquisite specificity. This characteristic allows monoclonal antibodies to be used as ...the DSHB, play an important role in scientific exchange. In addition to ensuring affordable access to ...

...the RNAi trigger delivering the RNAi trigger characterizing the RNAi phenotype. Although all these steps...plants to have enough tissue for downstream characterization. To simultaneously address both limitations...the RNAi output and trivializes downstream characterization, as the entire culture can be harvested instead...Stainier DYR (2020) Transcriptional adaptation: a mechanism underlying genetic robustness. Development 147....186452 Wilson RC, Doudna JA (2013) Molecular Mechanisms of RNA Interference. Annu Rev Biophys 42:217–...

...kinase activity is FRET, but FRET reporters are challenging to design and difficult to multiplex. The Covert...which fluorophores can be multiplexed together. Challenging  to Design: FRET reporter activity depends on...and NES sequences, which have previously been characterized. See Box 1 in Kudo et al. for more details on...Resources on the Addgene Blog Learn about photoswitchable kinases Learn more about fluorescent biosensors...

...Act on information to make change whenever possible. Be public about changes influenced by feedback. If... Human Dynamics Laboratory identified five characteristics of very successful teams. Check out Joanne'... post in this series). Members carry on back-channel or side conversations within the team. We are ...

...the lines between science and art was Albert G. Richards, who passed away in 2008. His work exposes and... use of x-rays. His haunting images have been enchanting people for years and have gathered much acclaim...Smithsonian Magazine, where the editor stated “Albert G. Richards, a teacher of dental radiography, had focused ...which are now extinct. A collection of Albert G. Richards’ work is held by The Museum of Jurassic Technology...

.... “When PhD students graduate there has to be champagne, that's a given.” Furthermore, anyone leaving ...fellowships, jobs, or paper submissions with dinner and champagne in the lab. The Church Lab celebrates successful...toasts a newly minted PhD with food, cake, and champagne. What does your lab do for fun as a group? The...those have been trips to breweries who happily exchange our expertise with lots of free beer.” He also...

...application process can dramatically increase the chances of being seriously considered for an open position...steps to submitting a resume that will give you a chance at an interview:  1) Have an excellent resume ready...or location. The more people you meet the better chance you have of finding a beneficial connection. LinkedIn...resume forwarded personally gives you a much better chance at an interview –but you still need to nail the...

...covalent bonds, so there were no weak links in the chain.  Addgene: What about the Streptococcus pyogenes...antibody tentacles may have an impact on this challenge either for magnetic capture or for capture using...this tool? Howarth: Polyproteins are useful for mechanical study of proteins by atomic force microscopy ...particular tips or tricks for those wishing to purchase and use the SpyLigase system for the first time...

... to students.” Making science approachable is often the most challenging part of the job, but Caitlin ...lab classes. However, their opinions are often changed after seeing a kit for themselves. “You open up...Center. And make sure to subscribe to the Career Channel of the blog so you don’t miss the future posts ...

...are ever recycled, so adopting a few meaningful changes in the lab could make a big difference in our global...Many scientists are already leading the cultural change toward responsibility in our lab’s environmental...Labconscious. If you’re inspired by this post then I challenge you to take to action! These days, it’s important...’s up to all of us to work on this issue. Be a champion for this important cause! To get more lab sustainability...

...appropriate antibiotic. Although the precise mechanistic details of the process are not well understood...resultant products of the reaction in the figure from Chaokun et al., 2011 to the right. After PCR, you should...with any PCR-based cloning technique there is a chance of introducing an unwanted mutation. Tips for high...(2016): 145-154. PubMed PMID: 27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free...

...40 cas gene families have been characterized. To date, 83% of archaeal and 45% of bacterial genomes utilize...defense against invading phages. Rising to the challenge - Evolution of anti-CRISPR genes in phages The...Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu S, Mojica FJM, Wolf YI, ...

...staining can be a challenge in the best of cases. But it can be particularly challenging when your only ...secondary antibody that is a Fab fragment, single-chain variable fragment (scFv), or a nanobody is one potential...Antibodies 101: Affinity Reagents Antibodies 101: Single-Chain Variable Fragment (scFvs) Resources on Addgene....

...difficult time. One function that rose to the challenge was our Finance Team. With Addgene closed for ...financial crisis. As a nonprofit organization, Addgene charges enough for our services to run our operations. ...to this need, we're incredibly grateful that the Chan Zuckerberg Initiative was able to step in to be ...laboratories all around the world." We thank the Chan Zuckerberg Initiative for supporting Addgene by ...

.... CRISPR 101: Homology Directed Repair Addgenie Chari Cortez explains some of the basic science behind...contributors David Wyatt and Dale Ramsden from UNC Chapel Hill explain some of the molecular gymnastics behind...Genome Edit Once you’ve used CRISPR/Cas9 to make a change to the genome, you need to check whether you’ve...for your next experiment? In this post, Addgenie Chari Cortez guides us through some common controls and...

... wanting to study bacterial genetics. This all changed thanks, in part, to a collaboration initiated at...Stanley Falkow, Stanley Cohen, Herbert Boyer, and Charles Brinton concocted a wild idea of using the newly...highlights of the 2nd edition eBook include: new chapters on genome engineering, cloning, and plasmid tags...Plasmids that Glow" and "Viral Expression Vectors" chapters, and more pictures and pro tips.  Happy reading...

...flexible work schedule the more we realize how casual chatter is very much missed in the virtual world. I believe...audience into small sessions and do a quick break to chat. This can be done randomly or manually assigning...could bring back a taste of that casual kitchen chatter, ultimately sparking new ideas. Social no-science-y...presentation will appear here, and only general chatter will be allowed with a different host each time...

..., the books center around a cast of endearing characters who decide to take matters into their own hands...the 1980s, Sarum follows an intriguing cast of characters from five different families over several millennia...one. On a purely selfish level, I wanted to be challenged to read some books outside of my comfort zone...s The Anthologist. The book ended up literally changing my life. Though I have an undergraduate degree...

...Associate Professor at University of Barcelona. The challenge: Making courses on genetic manipulation more hands...information they’ve absorbed if they don’t get a chance apply it in an interactive setting. The problem...could be played when and, depending upon who your challenger was, you might have to drastically alter your...please freely share your projects with us! This exchange of ideas will help improve life science and biomedical...

...complicated entities with a large range of characteristics (color, brightness, photostability, maturation...attach important references that serve to further characterize a protein of interest (and we very much appreciate...users to see when a given page has unmoderated changes and to view previous versions.  Currently, all ... plasmids Find plasmids for FRET Check out the Michael Davidson Fluorescent Protein Collection ...

...site. Cutting the linker by a caspase results in a change in fluorescence emission. FRET and fluorescence..., and thus quantify, due to small fluorescence changes of the donor and acceptor after proteolytic cleavage...fluorescence imaging of living organisms can be challenging due to issues with tissue autofluorescence, cell... cell heterogeneity, and/or cell position changes (To et al., 2016, Zhang et al., 2019). Thus to effectively...

...Skip to Chapter 3 for tips on how to develop your role as a lab leader, and then on to Chapter 4 for advice...the influence from his two PhD advisers who were “chalk-and-cheese”. While one was an established Professor....” Take home message Starting a lab can be a challenging process. Each PI emphasizes the importance of...

...our mission, it’s also because Addgenies love any chance to nerd out over their favorite science topics....Addgenie expertise we can. We even have a Slack channel just for asking people if they can write or review...posts get out of date as techniques and knowledge changes and require updating or retiring. CRISPR/Cas9, ..., for example, has gone through rapid change in the past few years. Thus, we are constantly updating our...

...available in your machine before you make your purchase. Picking a single fluorophore for an experiment...overlap, or the signal "spilling over" into another channel's detection range. Though you can compensate for... University Flow Core. Fluorochromes Brightness Chart. Boston University Flow Core. Retrieved January ...flow-cytometry/files/2013/06/Fluorochromes-Brightness-Chart.pdf Syracuse Flow Core. Syracuse flow core spectral...

...and γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (... vice versa. Envelope plasmids may be used interchangeably. Are γ-retroviruses replication incompetent...References and resources References Swift S, Lorens J, Achacoso P, Nolan GP. Rapid production of retroviruses ...cell-based systems. Curr Protoc Immunol. 2001 May;Chapter 10:Unit 10.17C. doi: 10.1002/0471142735.im1017cs31...