...disease characterized by increased iron serum levels and early-onset cataracts, is characterized by point...want to study RNA-protein interactions? The characterization of RNA-protein interactions has lagged behind...bind an RNA motif of interest in living cells. Challenges with existing RNA-protein interaction methods...containing three well conserved but not well characterized RNA motifs. This bait identified a new interaction...Ramanathan et al. found evidence that suggests changes in RC2H1 protein expression and post-transcriptional...Response Element (IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding...

...doi.org/10.1038/nmeth.3312 Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, Haque...for each loci, CRISPR/Cas systems only require changing the programmable guide RNA. Gene activation by...Tanenbaum et al., 2014, Konermann et al., 2015, Chavez et al., 2015). Most popular methods for CRISPR...other popular activation methods (VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using...method is its construction: it relies on antibody chains, which are relatively large and are not expressed...:7429–7437 . https://doi.org/10.1093/nar/gkt520 Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R...

... unpredicted gene expression changes (Sapozhnikov and Szyf 2021, Charlton et al. 2020). Most importantly... such as dCas9-TET, can, like the iron ball-and-chain that they resemble, actually impede your investigation... Figure 1: Break free from the ball and chain!   Epigenetics nation stuck with methylation...methylation state causes a condition. To understand mechanisms and, eventually, develop treatments, we need ...Read our CRISPR Guide to Epigenetics References Charlton J, Jung EJ, Mattei AL et al. (2020) TETs compete... https://doi.org/10.1038/301089a0. Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking...

...stable cell line. The genes encode the heavy chain and light chain for the antibody and when translated into...Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid...the line and subsequent cloning of the antibody chains (Crosnier et al., 2010; Andrews et al., 2019). ...reagents. These include conventional heavy and light chain recombinant monoclonal antibodies as well as other...With molecularly defined antibodies that do not change over time, scientists will know exactly what antibody...

...systems tend to have slower, but larger, calcium changes. The GCaMP6 family of sensors are commonly used...compartments can vary greatly. Additionally, the calcium changes you will observe in your experiment could be subtle...rearrangements of the sensor, and (3) subsequent change in fluorescence only occurs at high calcium concentrations...are available to simultaneously measure calcium changes in the ER, cytosol and mitochondria), the Cameleon-based...ratiometric GECO sensor (GEX), where one wavelength changes with Ca while another wavelength is insensitive...sensors have also been re-engineered to measure changes in calcium specific to the neuronal cell body or... Shen Y, Kramer L, Campbell RE. Structure-and mechanism-guided design of single fluorescent protein-based...

.... For example, the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed...of the patch cable (although you may be able to purchase one pre-made instead). Laser stimulation     ...      In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a mouse...determine the ideal excitation). Lasers can be purchased through Laserglow, which caters to optogenetics...PMID: 22157972. 3. Britt JP, et al. Use of channelrhodopsin for activation of CNS neurons. Current protocols...editorial board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. ...

....1021/acssynbio.6b00337. Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart...the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different modal...and Plant Parts Kit III (Gantner et al., 2018; Chamness et al., 2023). The Plant Parts II kit has a focus...for both monocots and dicots (Hahn et al., 2020; Chamness et al., 2023; Stuttmann et al., 2021; Grützner...elements such as promoters or fluorophores are interchangeable across organisms. When using the MK system,...Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12,... https://doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit...

...labeling reagents and structurally characterizing large ion channels involved in skeletal muscle contraction...Saraheimo et al., 2013), and visualizing structural changes in the ribosome during protein translation (Ermolenko...protein mass into your protein and so structural changes due to this bulky insertion are inevitable. Most...However, since tRNA suppression competes with natural chain termination at stop codons, expression levels of...with plasma membrane proteins (such as GPCRs, ion channels, or cell surface receptors) or with experiments...Figure 2). These labeling methods are freely interchangeable and new uses for FRET are continually being.... Kapanidis, Achillefs N., Yon W. Ebright, and Richard H. Ebright. "Site-specific incorporation of fluorescent...

...conjugate, and changing your reporter molecule requires either re-conjugating or re-purchasing your primary...have to be re-validated each time a new lot is purchased. Monoclonal antibodies, on the other hand, may...to switch from one readout method to another or change primary antibodies as needed. However, more antibodies...background noise as well, and multiplexing is challenging and limited. Direct westerns typically rely on...starting from tissue, you’ll likely want to use a mechanical method before your enzyme lysis. For cell cultures...you have an expert around, you’ll likely want to purchase one. If you need a single-percentage gel, you ...assumption that a specific protein’s expression has not changed due to ‌experimental conditions. You then normalize...

...anywhere.  Chapter 4 - JEI's mission and how they assess whether or not their achieving it. Chapter 5 - JEI's...Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing...Ho-Shing, and the Journal of Emerging Investigators. Chapter 2 - Getting high school students and teachers interested...the manuscript review/submission process at JEI. Chapter 3 - JEI target audience and their current readership...and developing one-on-one mentorship programs. Chapter 6 and Outro - Interview with high school student...

...because it contains two large heavy chains and two small light chains that stick out to form the “arms” ...access to routinely, and it can be especially challenging when trying to develop antibodies against unstable... derived from a camelid antibody called a heavy chain antibody (HCab). Because of their small size, nanobodies... phagemid pDX_init plasmid, using the fragment exchange (FX) cloning technique which uses Type IIS restriction...production of tag-free proteins for further characterization and analysis. In a rapid response to the COVID...Versatile High-Throughput Cloning System for Characterization of Enzyme Variants. In: Methods in Molecular...

...QuantificationPolymerase Chain Reaction Overview and Applications Resources on Addgene.org Protocol: Polymerase Chain Reaction...92. PMID: 19052298. Muhamad P, Chaijaroenkul W, Congpuong K, Na-Bangchang K. SYBR Green I and TaqMan quantitative...available, ddPCR may be a better option for more challenging cases.  Price point If budget is a concern...quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium.... PMID: 21554069. Haridan, U. S., Mokhtar, U., Machado, L. R., Abdul Aziz, A. T., Shueb, R. H., Zaid, ...

...oligomerize, protein charge, and actual molecular size – it is possible that a large or charged FP fusion can...also provide interesting insights into disease mechanisms and protein function. Getting ready to determine...Therefore, it can be beneficial to employ well-characterized marker proteins tagged with FPs to highlight...incorrect detection of one fluorescent signal in a channel setup for a different fluorescent signal) and photobleaching...can change the subcellular localization of a protein of interest. Scientists are continuously updating ...Endogenous Gene Tagging in Human Cells Check Out the Michael Davidson Fluorescent Protein Collection Get Tips...

...technologies currently available, you now face the challenge of trying to figure out what system might work... also consider FastCloning, first described by Chaokun Li and colleagues. Here you simply PCR-amplify ...overhangs left over from the PCR process (the precise mechanism isn’t well understood). Finally, TOPO cloning ...assembly can cost over $15.00 per reaction if you purchase a pre-made mix. Nonetheless, Gibson assembly is...efficient. Gateway cloning also offers a very high chance of success (>95% for a single insert), mostly due...finished constructs at Addgene!). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free... Michael G. Lemieux...

...contractions, and the control of voltage gated ion channels all depend on transmitting and sensing electrical...phenomenon you will likely want to monitor the voltage change across the membrane of cardiac cells. Similarly... out by action potentials which are electrical charges propagated through multiple cells to do things ...classified by their fluorescent reporter and the mechanism of their voltage sensing component, which separates...a variety of sources – voltage-gated potassium channels found in Drosophila to phosphatases from sea squirts...binding a Janelia Fluor (JF). The voltage-dependent changes in rhodopsin modulate the degree of quenching of...vectors become invaluable. Viral vectors are mechanisms by which genetic components can be delivered ...

...universities change their format of interview from a travel day to an online call in response to challenges on ...program itself. Picking a mentor comes with many challenges, and the flood of interview requests and visitation...standards for hybrid, virtual, and in-person events change seemingly daily (and are inconsistent between universities...idea is that the online meeting will give you a chance to get to know the faculty that you might be working...on their end. Though the quick changes in plans was stressful in and of itself, the flexibility allowed...everyone involved in the process. COVID-19 has changed many aspects of academia, and the PhD application...

...reversibly switchable (e.g. rsEGFP, Dreiklang). Fluorescent Timers (FT): These proteins change their color...Sensors: These FPs change their excitation/emission behavior upon environmental changes (e.g. pH, Ca2+ flux.../em spectra (e.g. mKeima) or change fluorescent intensity upon pH changes (e.g. pHluorin, pHTomato). ...wavelength. This means that the emission wavelength can change. In a few cases, the initial state of the protein...

...earlier this year by the White House as a Champion of Change, a program created as part of President Barack...Collection along with a BIOFAB kit comprised of well-characterized bacterial transcription and translation initiation...entirety of a cell. That's a big long-term technical challenge. We have a strategy that organizes how we start...engineered. For example, Boole’s logic shows up in mechanical systems, plumbing, computers, and now through...We know from our testing of these parts that the chance your genes will be expressed at the level you want...rebuilding existing genomes.) BIOFAB is laying down a challenge to its users and to the design community: If you...

...generating the standard curve for qPCR can be challenging to get right. Here at Addgene, we use a linearized...droplet generation oil are moved through small channels to create a water-in-oil emulsion containing approximately...factor of virus Most droplet readers have a few channels for detecting fluorescence so that it is possible... you should only need to use one. However, two channels can be used to examine the integrity of the viral.../s is recommended to ensure an even temperature change among all of the droplets, but you can go as low...droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis." Emerging microbes ...on the Addgene blog Read more about polymerase chain reaction Browse our blog posts related to viral ...

... Biology (Kimball et al., 2020). Benefits and Challenges CUREs inspire students to take ownership of their...research projects have also been proposed as a mechanism for increasing diversity and inclusion in STEM...experience on their resumes). CUREs are not without challenges. Real research is messy, and it can be difficult...instructors, and provides opportunities for students to exchange ideas and results with peers and faculty at other...win a Nobel!   Jason Pellettieri is Professor and Chair of the Biology Department at Keene State College...(4):602-606. Brian, D. 1996, Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  Callahan...Part I – guided inquiry – purification and characterization of a fusion protein: Histidine tag, malate...