...region. Two immunoglobulin (Ig) heavy chains and two Ig light chains make up each antibody molecule. The...the Ig heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable...diagram of an antibody including Fc, Fab, heavy chain, light chain, constant region, variable region   The... Nanobodies are the variable heavy chain fragment of heavy chain camelid antibodies (Hcab) (Arbabi-Ghahroudi...variable region of the heavy and light chain determine what antigen the antibody recognizes and binds to. ...The constant region of the heavy chain determines the antibody’s isotype - that is, one of five broad ...for specificity and sensitivity, for example by changing certain amino acids to improve its stability. ...

...Follow Jo on Twitter @JM_Grants and follow Grantchat @Grant_Chat    Additional Resources Read other Career...seekers to use online applications with significant character limits for the written portion of the grant application...government grantmakers. We keep up-to-date on the changing guidelines and regulations impacting grant management...degrees, foundations, coaching and even Twitter Chats dedicated to the Grant Profession.  Demand for grant...government. Throughout these moves and program changes, I learned to write grants for health, housing,...within the city infrastructure. My career path changed when I was asked if I would consider consulting... started my own grant consulting firm, a Twitter Chat, and then our grant training and mentoring. How ...

...proteins change configurations or binding partners, the epitopes available for binding change as well....often change shapes and epitope accessibility naturally, by binding to other proteins, changing locations...well. If this change hides the epitope that the antibodies used to recognize the protein, your antibody will... you're working with antibodies.) Conformation changes usually fall into one of two categories: ones induced...  First, let’s think about induced conformation changes. Several types of assays, such as ELISAs and Westerns... some of the bonds keeping the protein folded, changing it from its native state to a less folded, or ...and a more accurate separation of proteins by mass:charge ratio in an SDS-page gel. As the protein unfolds...

...family of plasmids by offering a lentivirus-based mechanism for sgRNA delivery and providing a means for large...mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to engineer a wide...Branchiostoma floridae, named bfloGFPs. They went on to characterize the structural and spectral properties of two...a mutant of the fluorescent protein dsRed that changes irreversibly its color from green to red when it... show its usefulness in cell culture to report changes in mitochondrial turnover and transport. pMitoTimer...degradation in vitro and in vivo. Merged green and red channel fluorescence images of C2C12 cells stably expressing...alternative pathway or induce other compensatory mechanisms. How can protein function be dissected while ...

...) are fluorescent proteins that display unique changes in their spectral properties upon exposure to a...fluorescent states to high fluorescence states, they can change from one fluorescent color to another, or their...Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission...usually preferred for studying single protein characteristics, as the monomeric state is less likely to interfere.... By generating monomeric PA-FPs, there is less chance that the chimeric protein will oligomerize, disrupting...super-resolution imaging usually have the following characteristics-high contrast ratios, bright fluorescence, ...

...a decade of frenzied interest in bacterial and archaeal immune systems. During this time, scientists discovered... discovered many related immune mechanisms that could target foreign DNA with unique properties. This ...fast evolution of these adaptive bacterial and archaeal immune systems has led to extreme sequence divergence...share sequence similarity but are grouped due to mechanisms of action and other analyses. This also means...identified CRISPR-Cas in bacteria and nearly 100% in archaea, though Class 1 remains largely underutilized compared... These subtypes have similar compositions and mechanisms, with the biggest differences being the components... contain a distinct Cas7-type gene. The exact mechanism of type IV systems is not known, but it’s suspected...

... that changes color, but the experience is invaluable. This opportunity has given us the chance to develop... careers in order to head to Ireland and take a chance as entrepreneurs. To make things more interesting...product no one has ever seen before (flowers that change color throughout the day) and doing this with very...our lives and took this daring risk? What moment changed our whole outlook on the possibilities in life ...published a paper a year (or less) and limited the exchange of information to the public and to other academic... publish or attend conferences makes it very challenging to compete in today’s tough academic or industry...team. We have confidence that we can take on any challenge and quickly become experts in something, even ...

...science publishing system needs to be changed.  Dr. Bourne charged the audience to use their useful energy...things off, “We are all capable of contributing to change.”  Check out Joanne's Reddit AMA The keynote speaker...spoke eloquently and vehemently about the need for change.  Dr. Bourne strongly believes this is an amazing...PI driven basic research, there is resistance to change, public support is weak, government support is ...is “feckless”.  So what can be done?  He charged the postdocs to “Grab ahold of your destinies” and also...engaged in training excellence to begin effecting change.  Panelists call for action Each of the speakers...graduate students and postdocs in their labs. Dr. Richard Roberts, CSO of New England Biolabs, gave examples...

...3b in Chan et al.   To achieve the full diversity of colors AND sparse labeling of neurons , Chan et al...both the CNS (figure 5 in Chan et al) and the PNS (supplemental figure 8 of Chan et al). These new AAVs ...figure 5 and supplemental figure 8 in Chan et al).  References 1. Chan, KY, Jang MJ, Yoo BB, et al. Engineered...3) they have limited toxicity. However, a key challenge of using AAVs for neuroscience research is the...the Gradinaru Lab at Caltech stepped up to the challenge. Find the PHP Plasmids Here! Their Solution: ...the cell type of interest, which, in the case of Chan et al, was neurons and astrocytes. Why screen for...high doses of this viral cocktail are injected, by chance lots of individual cells will be transduced with...

...transport suggest that there are several parallel mechanisms in place (for review see Maday et al., 2014)....neuroscientists for some time due to their advantageous characteristics for brain research and their ability to introduce...2010), AAV8, -9 (Masamizu et al., 2011); AAV5 (Aschauer et al., 2013). Anterograde transport has been ...California Institute of Technology generated novel AAVs (Chan et al., 2017) that express one of a few different...are capable of synaptic spread. The underlying mechanisms of trans-synaptic trafficking are not fully understood...these two modes of transport may share common mechanisms. Rare observations like these may be a starting...brain. A better understanding of the routes and mechanisms of intracellular transport of AAV could facilitate...

...Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain fusion...Can be used for in vivo models Single-chain Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV ...Bratovič M, Charpentier E, Möglich A (2013) Engineering of temperature- and light-switchable Cas9 variants...Y, Liu Y, Qi LS, Lin MZ (2017) A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible...Moritoshi Sato’s lab at the University of Tokyo and Charles Gersbach’s lab at Duke University developed similar...Their most successful design utilized Magnet photoswitchable proteins derived from the fungal photoreceptor...instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system’s efficiency...

...work in Michael Lin’s lab at Stanford, Xin Zhou greatly expanded that list by using photoswitchable fluorescent...apply fluorescent proteins everyday.  The late Michael Davidson and Roger Tsien played enormous roles ...To honor their legacy, we recently launched the Michael Davidson and Roger Tsien Commemorative Travel Awards...this post, we’re proud to announce our first two Michael Davidson and Roger Tsien Commemorative Travel Awardees...turned the enzymes back on. Xin termed these photoswitchable enzymes Fluorescent Light-inducible Proteins...various designs, Xin has been able to develop photoswitchable Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5,... You can learn more about Xin’s work with photoswitchable components of the ERK signaling pathway in ...

...ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one of the repair...template for repair and is thus the more accurate mechanism for DSB repair. The process is generally error-free...the template DNA. There are several unique HDR mechanisms, but all templated-repair pathways have these...process Homology directed repair pathways HDR mechanisms can be divided into two main camps: non-conservative... conservative. The conservative methods are characterized by use of a homologous donor (e.g., sister chromatid...single strand annealing (SSA), an error prone mechanism that notably does not require a donor template... (SDSA) pathway SDSA is a conservative repair mechanism which exclusively yields non-crossover events....

...founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process of founding...ensuring that success Chapter 6 - Developing Addgene’s Brand and culture Chapter 7 - Building Addgene ...distribution!) Chapter 8 - Opportunities to create repositories with other materials Chapter 9 - The Founders... came from and how the idea got off the ground Chapter 3 - Addgene as a leader in standardized tech transfer...transfer and the adoption of the UBMTA Chapter 4 - Developing Addgene as a modern e-commerce company and... and establishing credibility Chapter 5 - Realizing that Addgene would be successful and the importance...and beyond - Ginkgo, Tufts Launchpad, and more Chapter 10 - Addgene's Future, the research team, and the...

...jellyfish Cassiopea xamachana. Marine Biology 94:423–430 . https://doi.org/10.1007/bf00428249 Chan MCY, Karasawa...-colored protein from the jellyfish Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years later...of chromoproteins were identified in sea anemone (Chan et al., 2006). Why do these organisms have chromoproteins...specific purposes.     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP... create negative selection markers for allelic exchange. These vectors contain a toxin gene (regulated...rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange occurs through two sequential homologous recombination... the tsPurple chromoprotein.  Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET acceptor...

...identify how MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced... Entropy as a Proposed Gating Mechanism for Magnetogenetic Ion Channels. Biophysical Journal, 116(3), ... good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet was passed ...Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT magnet. Gray regions indicate...could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool, ... Q513K, G528K, D540M) that shows a fluorescence change of ~75% in response to the magnetic field (Figure...MagLOV-based tools, just as light-induced conformational changes are the basis of many optogenetic tools. And, since...

...flank the desired change with 4-5 silent changes in the wobble codons, i.e. make changes to the third base... translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds- ...sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has ...to restriction enzyme cloning where the genetic changes occur in a test tube. The donor DNA substrate only...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...prone to mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that... to generate insert ssDNA Point mutations or changing a few nucleotides 0.1-1% or 25-50% when activation...

...bacteriorhodopsins, halorhodopsins, and channelrhodopsins, the first two found in the Archaea, the third in green algae...). These proteins control the flow of electric charges across cell membranes and maintain membrane potential... 2012). For example, blue light activates channelrhodopsins that, in turn, trigger neural excitation, ... promoter upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry...vectors to study the neural circuitry behind the mechanisms of sleep and anesthesia. In Cre transgenic mice...vectors to answer this question, mapping and characterizing the activity of the DA neurons of the VTA. ...optogenetics to understand the cellular and molecular mechanisms of the brain in vivo. Many thanks to our guest...

...this is not a direct reference to the video game character but rather to an extremely important gene in animal...1980, when Christine Nusslein-Volhard and Eric Wiechaus first identified the hedgehog gene in Drosophila...that resembled a hedgehog (Nusslein-Volhard and Wiechaus, 1980). The mammalian hedgehog protein, sonic ...into vertebrate chromosomes via a cut and paste mechanism. Scientists reconstructed a transposase system...squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess et al., 1991...plasmids in our inventory from the lab of Eric Wieschaus that express sqh.   cookie monster, a meiotic...required for spermatogenesis after a Sesame Street character? The scientists who discovered comr thought the...

...constant domains, the plasmids for the heavy chain and light chain are also available from Addgene!   ...disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody collection grows...K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies to simian virus 5...of its biology, pathogenicity, and resistance mechanisms.     Figure 4. Overview of the dual-plasmid...10.1128/jb.00335-23.  Bioluminescent reporting of mechanical forces with PILATeS By Emily P. Bentley Time ... lab and collaborators have deposited a new mechanosensor called PILATeS, Pulling-Induced Luciferase Association...different contexts.   Figure 5: Left: Mechanism of the PILATeS tension sensor. The LgBiT (cyan...