... microscopy. Nanobodies can be labeled with up to 3 dyes, yielding similar signal strength to a standard...nanobodies are useful for colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary...PMID: 26633879 PubMed Central PMCID: PMC4755751   3. Muyldermans, Serge. “Nanobodies: natural single-domain...Control Protein Activity Learn more about protein tags Read about E. coli strains for protein expression..., if not better, than antibodies for all of the above listed molecular techniques, but they can also be...protein purification methods. Read on to learn more about nanobodies and how their structure and function ...between a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-resolution...

...selection techniques to come up with new colors (Fig. 3), such as Radish and Carrot, and further means to ...imaging in situ”. Nature Methods 9, 787–790 (2012). 3. Filonov, Gregory S., et al. “In-gel imaging of RNA...RNA-Modifying Enzymes.” Cell Chem Biol. 2016 Mar 17;23(3):415-25. PubMed PMID: 26877022. PubMed Central PMCID...target metabolite creates an aptamer that will only bind to DFHBI in the presence of that metabolite (Fig...approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing...fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the...Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein Activity with GFP Resources...

...effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid B (as positive control...we have successfully transfected our cells (Figure 3), we can continue with our experiment, incorporating...optimization within your experiment. Read on to learn more about the various controls that can be used for plasmid-based...: Expression Level of Gene X The results shown above are from a single experiment wherein Plasmid A (...be compared. The Empty Vector Control (mentioned above) could also serve as an important negative control...experimental cells. For example, in the experiment above, Plasmid A contains an shRNA that targets human ... times and on different days. Using our example above, we would test different preparations of Plasmid...

...possible because there are 4 bases (A, T, C, and G) and 3 positions in each codon. There are therefore 64 possible...possible codons but only 20 amino acids and 3 stop codons to encode leaving 41 codons unaccounted for....PMID: 15678157. PubMed Central PMCID: PMC1299251. 3. Emilsson, Valur, and Charles G. Kurland. "Growth ...that lead to these preferences and what can we do about them? Read on to find out!   Why do organisms have...understood, but some possible reasons include: Metabolic pressures - it takes cellular resources to produce...amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...Effects on transcription (remember, it’s not all about translation - e.g. codon choice may interrupt transcription...

... than fusions to 3 or more HA tags. 4. High-resolutions microscopy As indicated above, smFPs can be used...moleculare were better resolved with smFP_FLAG than GFP.   3. Enhanced ability to distinguish weakly expressed ...brain". Oxford University Press, New York. pp 719. 3.Rizzo, Mark A., Michael W. Davidson, and David W. ...enable accurate labeling of neurons. As discussed above, weakly expressed proteins in particular are difficult...resembles images of the deity (see comparison image above). The church of the Flying Spaghetti Monster is ...

...fidelity of cohesive end ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction...assemblies and over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases...plate using outgrowth volumes described in Figure 3, with calculated yields from entire outgrowth built...Inserts, either precloned or in amplicon form (shown above) are flanked by BsaI sites in an orientation that...Check out NEB's work on ligase efficiency Learn about other plasmid cloning techniques Resources on Addgene.org...

... different systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion ...activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including many...Science. 1986; 231:699-704. PubMed PMID: 3080805. 3. Brent, R., and Ptashne, M. A eukaryotic transcriptional... Plasmids Read our Blog Post About Yeast Vectors Read our Blog Post About Tagging Yeast Genes ...

...program to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination ...Gateway cloning efficiency can reach >99%.   STEP 3: Express your Gene of Interest! Be sure to verify ...2003 Feb;Chapter 5:Unit 5.17. PubMed PMID:18429245. 3. Ptashne, M. (1992). A Genetic Switch: Phage (Lambda... expression systems. Keep reading to learn more about the Gateway cloning method and its advantages. An... on the Addgene Blog Try Out FastCloning Learn About Methylation and Restriction Enzymes Use Colony PCR...

...genome which was composed of the wild-type 5’ and 3’ LTRs, the ψ sequence, a part of the env gene containing...exception of the env gene (packaging plasmid), and 3) the vesicular stomatitis virus G protein (VSV-G) ... been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the best production...Science 272.5259 (1996): 263. PubMed PMID: 8602510. 3. Yasutsugu Suzuki and Youichi Suzuki (2011). Gene ...first one and can be used routinely in a research laboratory. The risk of generating RCL is low but extra ...even your own! You should therefore be cautious about inserting potentially harmful genes into a transfer...

...sequence for recognition by QCascade; although 5’-CC-3’ is one of the best PAMs for VchINT, many other PAMs..., by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple distinct targets... or isolated (Rubin et al., 2020).  Figure 3: Multiplexed insertions with Type I-F INTEGRATE using..., 2020), to multi-copy genomic integration of metabolic cassettes (Zhang et al. 2020); however, the technology...

...improve AAV yields. Although we only investigated 3 serotypes it’s possible that this approach will be...bioengineering 42.5 (1993): 601-610. PubMed PMID: 18613082. 3. Oyaas, K., et al. "Hybridoma growth and antibody ...resources on Addgene.org Learn about Addgene's viral service Learn about viral production Find viral protocols... By now you have probably heard about Addgene’s viral service but did you know that Addgene also performs... approach can be adapted by vector cores and laboratories alike as an easy and inexpensive method to improve...Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production." Biotechnology and ...

...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and ...Connie Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this... answers to the top questions scientists ask me about mentoring. Here they are: What makes a good Mentor... Where can I find a Mentor? What is this I hear about “group” or “peer” mentoring? How do I ask someone...a good science mentor? I started a conversation about many of these topics with the researchers who joined...often one that devotes time and energy to thinking about how to be a great Mentor.    Check Out Angela Depace's...questions in future posts. If you have a question about mentoring that you want us to cover, let us know...

...Resources on the Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve HDR Efficiency How...nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design.  Exploring nickases...

...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies ...us know. We’d love to hear from you!   Fig. 3: Image of Addgene’s western blot for the myc-tagged...Brian O'Neill  The François St-Pierre lab and collaborators have successfully developed an improved voltage...

...depending on the needs of your experiment (Figure 3). And using self-labeling tags makes updating your...ligands at different time points.   Figure 3: Single-molecule tracking of actin filament assembly-regulating...Imaging and Protein Analysis. ACS Chemical Biology, 3(6), 373–382. https://doi.org/10.1021/cb800025k Miller...Article 1. https://doi.org/10.1038/s41467-023-39655-3 Wilhelm, J., Kühn, S., Tarnawski, M., Gotthard, G....but it’s real! In this post, we’re going to talk about the power and flexibility of self-labeling protein...ready-to-use without any cloning at all! As mentioned above, several variants of these tags have been developed...Illuminating Choices blog post for more discussion about choosing a dye or ligand to use for your experiment...

...series of one- or two-marker plots, like in Figure 3 above, until the population of interest has been successfully...location of the cell populations as a guide  (Figure 3.) You can see that in our dot plot, we have A-B- cells... but no cells that are A+B-.      Figure 3: It's often easier to identify different populations...  Of course, there are many more things to learn about when diving into flow cytometry data. There’s different... ways to use FSC and SSC, as briefly alluded to above. There’s many variations in how one can gate, select...

...evidence you need. For example, as shown in this study [3], hippocampal neurons are activated when the mouse...PMID: 25242321. Pubmed Central PMCID: PMC4181379. 3. Kheirbek, Mazen A., et al. "Differential control ...retrieval strategies for remote memories. Cell. 2011;147(3):678-89. Pubmed PMID: 22179551. Pubmed Central PMCID...of the important considerations you should think about when developing optogenetics behavioral experiments...scientific research is that the field benefits from collaboration and the exchange of ideas. The rapid spread ...versatility and standardization of genetic tools in the laboratory mouse, most optogenetics is performed in this...

...bind to the anti-Protein B antibody (Fig. 3)    Figure 3: Recommended secondary antibodies for rat...flexibility needed for multiplexing. In the case above, you can’t costain with your desired antibodies ...multiplexing with chimeric antibodies. In the case above you should use an anti-rat IgG1 secondary antibody...this blog post has answered all of your questions about the utility of isotype conversions and chimeric ..., Bright, S. M., Robinson, M. K., Graham, E. T., Sabo, J. P., Owens, R., King, D. J., Norris, S. H., &...

...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth...Heppert JK, Higgins CD, Goldstein B. Genetics. 2015 Jun 3. pii: genetics.115.178335. [Epub ahead of print] PubMed... this species, namely, the robust HDR mentioned above, an accessible germline, and multiple selectable...modular system that requires very little hands-on labor and could facilitate the tagging of every protein...

...decided to pack it in. I had just started month 8 of my 3 year Post-Doctoral contract at the University of Cambridge...this job specifically looked for a researcher with 3 years+ of cancer research experience, a PhD or a Master...for Dummies”. The fastest way to learn something about marketing would be to start reading a broad simple...introduction to the financial world. It teaches you about selling products which is an important part of the...Seán on twitter @Macfhearraigh.    Read more posts about Scientific Career Development ...