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...combined in one step. As with standard IF, you will need to start with a fixed sample. Depending on... antibodies. The general process is similar to standard immunofluorescence, but with some extra considerations...photobleaching or by stripping out the antibodies, then start over with the next target: add the new primary, ... will need to include a marker like the Hoechst stain for the nuclei. (See Brewer et al, 2022 for a sample...Abcam's sample protocol is a good place to get started.  One important consideration when deciding whether...magenta), anti-AnkyrinG (green), and Hoechst nuclear stain (cyan). Panels C1-C3 show magnified detail of the..., many of the same considerations apply as for standard IF. However, there are a few specific considerations...

...forget to start it, the samples can actually migrate into the buffer — so it’s better to start over if ...consider using an unstained protein ladder, which will run a bit more true to size than a stained ladder. Unstained...transfer worked with Ponceau S staining or other reversible protein staining immediately after your transfer... Check transfer with Ponceau red stain or other reversible stain* Roll out all of the bubbles in...dilutions of your sample or protein of interest to understand the dynamic or working range of a given reagent... runs to optimize or troubleshoot.   Before you start… Mark your membrane! Membranes look the same from... amounts of protein, make sure your reversible staining method of choice is appropriately sensitive. If...

...time, plasmid prepping is a breeze. You get your stab from Addgene, streak for single colonies, sub-culture... genotype, as these mutations improve plasmid stability and yield. If a plasmid is prone to recombination...plasmids with inverted terminal repeats. We use NEB Stable to transform and prep high quality DNA that contains...recombination, but are also endA+. endA encodes a thermostable periplasmic endonuclease that can sometimes ...of common E. coli strains. Increase the volume Starting with a larger culture volume can improve plasmid...performing filter based plasmid prepping. Be sure to stay within the recommended range of the filter or you...growth conditions. For many high copy plasmids, standard LB broth and antibiotic concentrations will work...

...of preference, and it will be stable for years. Plasmid DNA can be stable at 4°C or even room temperature...answer: yes. This last step will influence the stability of your sample over time and determine which experiments...effectively use the DNA. pH and DNases affect DNA stability Figure 1: So many choices! What to do? ... The main factors that can affect DNA stability in solution are pH and DNase contamination.  Extreme... favors the breakage of the hydrogen bonds, destabilizing the molecule and favoring the separation of ... be able to store your sample without having substantial degradation and which experiments you can use... buffer is the best buffer for preserving the stability of your preparation for a long time. Tris buffer...

...office is a good place to start. It’s not unusual to find science writers who started out as graduate students...skills. So, where can a graduate student or post-doc start? The good news is there are more opportunities than...Here are my recommendations on some ways to get started sharing your science with the community. Some of...father or your best friend who isn’t a scientist, start. If they don’t seem all that interested in the details... You’ll know you are on the right track if they start asking you questions. Remember that people like ...often, failure. It’s useful for non-scientists to understand not just the details of your science but also...written by practicing scientists. 3)   Consider starting up your own twitter feed or talking about and ...

...have only been stained with secondary antibodies, showing the level of background staining. All antibodies... orthogonal approach to understand the relationship between antibody staining intensity by flow cytometry...different depending on the sample and target type. To start the validation process, one must first confirm that...permeabilized in 0.1% saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and...CoraLite® Plus 647 secondary antibodies. Antibody staining was quantified using the Attune NxT Flow Cytometer...Cytometer with representative images showing the staining intensity in the KO population (pink histogram)...maximize the separation and reduce any background staining. This can include using: different blocking reagents...

.... Now GFP and its fluorescent derivatives are a staple in the lab. GFP is used in research across a vast...consisting of 238 amino acids derived from the crystal jellyfish Aequorea victoria. It has a fluorescent...that increased the fluorescence intensity and photostability of GFP. This also shifted its major excitation...to GFP and new iterations of fluorophores are constantly being engineered. Table 1 lists a few common ...Known Function S65T Increased fluorescence, photostability, and a shift of the major excitation peak to...Increases quantum yield for BFP Y145A and H148D Stabilizes the structure of Cerulean derivatives F99S,...GFP and other fluorescent proteins have become a mainstay in molecular biology. Scientists can easily utilize...

...products of teams: Watson, Crick, & Franklin, Meselson-Stahl, Hershey-Chase...Who was I to dispute history? And... I happily embrace (and admittedly, I may have started that nickname myself). I have my own little Product...shipped; Chiara, the tech transfer expert who understands the somewhat complicated Material Transfer Agreement... kit pages. No single person could possibly understand every nuance of how kits work at Addgene, but...and others, we have updated our kit process substantially and have plenty of ideas for further improvements...biochemist, a geneticist, a microscopist, a biostatistician, and that one golden-handed technician who ...Andrew, the team’s founder, to create a mission statement in the first few meetings and we’ve volunteered...

... “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting when I...chicory root, but considering this is a needle in a haystack type of objective, it was surprisingly easy. It... See below for details. The entire process from start to finish took about 1 month. Cas9 delivery ...particular experiments, we generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral...blotting was performed on whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing...with too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (.../mL blasticidin for 9 days. Single cells from a stable cell pool were then expanded for an additional ...

... Applied Plant Sciences at The Ohio State University. Established in 1991, the Arabidopsis Biological ...Arabidopsis and related species. Located at The Ohio State University in Columbus, Ohio, ABRC ships more than...Center (ABRC) set out to remedy that with the establishment of an outreach program in 2011. More than a...the Classroom, were developed and tested by ABRC staff. These kits provide in-depth, ready-to-teach lab...experiments allows them to develop a deeper understanding of scientific concepts than can be gained from...benefit of using Arabidopsis in the classroom is that staff from the ABRC are here to support you! From questions...guiding students through specific protocols, ABRC staff are available via e-mail and phone to answer questions...

...are crisp, bright, and lacking in non-specific staining can be a challenge in the best of cases. But it... antibody, resulting in significant background staining (Figure 1A). Not all tissues will have the same...researchers can get away with species on species staining with no problem! But to be sure that your secondary...experiments. If you do find high levels of background staining by the secondary, then the following three strategies...help!     Figure 1: Species on species staining can be a problem because the secondary antibody...that you can significantly decrease background staining by mixing your primary and secondary antibodies...are a few common solutions to avoid background staining when doing species on species IHC. (A) One solution...

...mCerulean3, mKOFP2) and empty vectors that have standard cloning sites or gateway compatible cloning sites...factors OCT3/4, SOX2, KLF4, and c-MYC is the established model for reprogramming somatic cells. A common...alkaline phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing...like transgenic mice are important tools for understanding the function of cell types in the nervous system...they constructed AAV plasmids bearing one of the distal-less homeobox 5 and 6 (Dlx5/6) enhancer elements...stokes shift, long fluorescence lifetime, and a stable monomeric form as determined by the organized smooth...mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet, fluorescent lifetime ...

...you as a colleague and give you a million dollar startup package. There are three steps to chalk talk preparation...what" question. For example, "we want a better understanding of the Z pathway" is vague and "what proteins...outcome makes it easier for the search committee to understand your goal, what you expect to find, and how you.... Finding evidence for or against a model is a standard scientific strategy. However, a robust research...Aims should have short titles, a clear easy-to-understand question/goal, an approach, anticipated outcomes...describing. It's easy to be overly ambitious – a common mistake is to make each aim into a 10-year research program...idea. It should be at a high level, broadly understandable to scientists outside your field, and interesting...

... remotely. Social distancing? Many people are now calling this “physical distancing” since it’s important...important to stay virtually connected with the people in your life. At Addgene, we’ve started virtual activities...keep calm and science on. Practice self-care and stay connected Find time for exercise. Many studios ...these are activities that you can easily do while staying away from others.  Meditate. Meditating during ...lunch, and sharing photos of our pets. #SocialDistancing may send a wrong message for some people. .... We should recommend #PhysicalDistancing. Social interactions and #social support are even more necessary...your work online. Maybe you’ve always wanted to start using social media professionally but haven’t had...

...scientist started with the Addgene blog. I wrote my first blog post out of boredom. I had just started working...wondering how to get started. This blog post outlines 10 steps to take when starting to use Twitter as a...Before I started writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media...social media professionally, and 2) I needed to start using Twitter. Sharing Chemistry Cat memes use...science It may have taken a bit of FOMO to get me started, but now I’m using social media to help my research...to do with it. Five resources to help you get started with social media as a scientist Using social media...it’s hard to know where to begin. To help others start using social media to their professional advantage...

...manager,” you’re not alone. A few years ago when I started my career transition from bench science to software...product manager works with internal or external stakeholders to learn more about their needs and any problems...trying to solve. After learning more about the stakeholders, the product managers collate and organize that...developers to help implement solutions for the stakeholders. Finally, software product managers test the...solutions with the users, gather more feedback, and start the cycle over again to refine the solution, or ... workflows into a format the developers could understand, so they could code the best operations software...have three or more concurrent projects in varying stages, which can get messy if you’re not on top of it...

...protonated and a deprotonated state (Tsien, 1998). The deprotonated state has an absorbance maximum around...protonated state, however, does not absorb light at 488 nm. The ratio between the two states is reflected...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...add a co-factor, (iii) easy visualization with standard filter sets on a fluorescence microscope, and ...N-terminus, but this hardly reduces its size. The substantial size of GFP may interfere with the activity or...specifically bind the molecule of interest. For instance, pleckstrin homology domains can be used to detect...2018). GFP is not tolerated in my fusion It is standard practice to attach the GFP to the N- or C-terminus...

...incorporation falls with increasing distance from the cut site. If the distance is only 10 bp, the efficiency...to mutation distance and editing efficiency.   3. Optimizing "cut-to-mutation distance" for homo or...re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore (3), we have developed a framework...CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though we had controlled over-active Cas9...phenomenon and found a general relationship between the distance of a mutation from the CRISPR/Cas9 cut site and...heterozygous mutations When thinking further about the distance relationship described above, we realized that...incorporation drops with increasing cut-to-mutation distance, the mutation may only be inserted at one of the...

..., the monomeric state of a probe leads to less interference with the oligomeric state of a fusion partner...Molecular brightness vs. iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells ....8 33 State-of-the-art NIR FPs, which combine high cellular brightness and monomeric state, are highlighted...there is a Table that summarizes the properties of state-of-the-art probes, many of which you can find at... heme metabolism (Shemetov et al, 2017). Modern state-of-the-art NIR FPs can be used similar to GFP-like... by an apoprotein and protein expression level. State-of-the-art FPs (listed in Table 1) were optimized...light-oxygen-voltage (LOV) domain. They can also be used in crosstalk-free three-color imaging with green and red GFP-like...

...C-terminal HA tag. Selectable and stable: A puromycin resistance gene makes stable cell line creation a breeze...step-by-step protocols for viral generation and stable cell line creation (Figure 2). Looking for more... plasmid set for expressing tagged proteins, and stay tuned for mammalian expression and AAV plasmid sets...supernatant and selected with puromycin. Puromycin-resistant cells were fixed and labeled with primary antibody...Alexa Fluor 594 (Thermo Fisher A-11012), and counterstained with DAPI. New recombinant antibodies...the payload sequence into the genome. The team established that they could reprogram the genomic target ...dynamic protein labeling By Alyssa Shepard To fully understand how genes are regulated, it's important to know...