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Showing: 121 - 150 of 846 results

  1. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    Published
    April 14, 2016, 2:30 p.m.
    ...Research Fellow with Pamela Silver.  Most types of biological motion (whether endocytosis, vesicle trafficking...it to suit our purposes? In search of simple biological machines R bodies can repeatedly extend and...R-bodies, check out this video. Most types of biological motion (whether endocytosis, vesicle trafficking...to pH changes without the need for ATP or other chemical fuel sources. Like an actin filament or a microtubule...polymers, however, R bodies do not exert force by dynamically rearranging into different polymerization states...body monomers respond to pH changes by undergoing local conformational changes. These tiny movements are...by propagating through an enormous lattice of identical subunits. A brief history of R bodies R bodies...

  2. Addgene Welcomes John Doench to the Advisory Board

    Type
    Blog Post
    Published
    Aug. 13, 2015, 2:30 p.m.
    ... has been critical to the Broad’s success over the last 11 years and was certainly critical to drawing...background in history and specifically the history of RNAi left him a bit skeptical of the technology at the...cancer biology in Ed Harlow’s group at Harvard Medical School and then applied to work at the Broad Institute...towards the Broad. In order to ask interesting biological questions, you need a tremendous amount of technology... in true collaboration with scientists asking biological questions. Doench loves that he still gets to...terms of determining what genes are involved in biological process X, you can do with CRISPR technology....with Addgene - he says he’s “known of Addgene basically since the beginning and [has] been using Addgene...

  3. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    Published
    April 13, 2023, 1:15 p.m.
    ... Clinical applications Viruses in vaccines helped pave the way for their use in other clinical applications...vector from basic science research all the way to clinical gene therapy. But how can you use these amazing...and delivering a genetic payload. This cargo is typically their own genome, but this step can be engineered...’ve decided on an application, our Viruses as Biological Tools blog can help you learn about the different... express proteins. Viral expression cassettes typically contain a promoter (inducible or constitutive)...delivery Screening libraries, large collections of biological independent variables, are becoming increasingly...development well over a hundred years ago. Since then, medical technology has changed a lot, but viruses have ...

  4. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    Published
    May 13, 2025, 1:15 p.m.
    ...into those of the cells they infect. These are typically DNA viruses, such as adeno-associated virus, herpesviruses...cycle to continue. They rely on a viral protein called reverse transcriptase to convert their RNA genomes...plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene encodes...encodes three separate enzymes that are proteolytically processed from the larger protein: PR (protease), RT...of lentiviral particles.  The envelope plasmid typically encodes the vesicular stomatitis virus G protein...could lead to an undesirable interruption of a critical gene. Because the integration of lentiviral vectors...Insertional mutagenesis is especially concerning in clinical settings, as evidenced by retroviral gene therapy...

  5. dTAG - You're it!

    Type
    Blog Post
    Published
    June 21, 2018, 2:06 p.m.
    ...Rather than inhibiting protein function, these so-called “small molecule degraders” recruit the proteasome...degraders provide distinct advantages over pharmacological inhibitors including rapidly depleting a protein-of-interest...resistance to inhibitors, and inducing prolonged biological effects. While targeted degradation of endogenous...selective degradation of protein targets in time scales that are not possible with traditional genetic ...here. The Methods section of Nabet et al., Nature Chemical Biology additionally provides detailed information...FKBP12F36V binding ligand (ortho-AP1867) via various chemical linkers. Our current lead dTAG molecule is dTAG...FKBP12F36V-tagged LACZ) with dTAG molecules in your biological assay of choice to ensure that there are no toxicities...

  6. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    Published
    July 25, 2017, 1:20 p.m.
    ...pathway (Raf1, EGRF), the KTR localized to the cytoplasm. This all-optical psMEK1 + ERK KTR-mRuby2 system...since other signaling networks can also be non-specifically activated and, like the game of telephone, often...must be transmitted by intermediate messengers. Optical control of kinase activity can provide greater ...greater spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist...Problem:  There is no common method for creating optically controlled kinases. The Solution: In Zhou et al...constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 nm violet light, the...phosphorylation, much like endogenous ERK, and its localization serves as an indicator of MEK1 activity. When...

  7. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    Published
    June 15, 2017, 2:30 p.m.
    ... 2016; Constantini et al, 2012). The localization and biological properties of new fusions can be compared...previous two posts in this series described a practical approach to selecting a bright fluorescent protein...ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity and has...efforts tested whether FP fusions to connexin localized correctly to gap junctions (Shaner et al, 2008... with native interactions, preventing proper localization in these assays. However, these strategies will...most likely in an antiparallel configuration), typical structures known as Organized Smooth Endoplasmic...the performance of fluorescent proteins in your biological system with the microscope that you will be using...

  8. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    Published
    April 12, 2016, 2:30 p.m.
    ...networks underlying your favorite biological phenotype in a scalable way. This blog post will introduce...by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts to study and ... guide RNAs (gRNAs) for genome editing in large-scale has led to the rapid generation of genome-wide gRNA...genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems for high-throughput... genetics that underlie complex regulation in biological systems. The CombiGEM platform provides a means...currently an Assistant Professor at the School of Biomedical Sciences of the University of Hong Kong. He is...technologies to interrogate and understand complex biological systems. He can be contacted at [email protected]. ...

  9. Adapting Toehold Switches to Detect Zika Virus

    Type
    Blog Post
    Published
    Aug. 30, 2016, 2:30 p.m.
    ...Organization has declared a global health emergency and called for the fast-tracked development of diagnostics...was a new class of programmable riboregulators, called toehold switches, which allow for the rational ...outbreak was reported, and we felt that, given the critical need for low-cost, field-based diagnostics, our...Our motivation was underscored by the potential scale of the emerging outbreak, as many as 2.2 billion...timeline to develop our paper-based platform into clinically relevant diagnostics for the Zika virus was daunting...ability to complete the project on such a short timescale. Our article ultimately reported the development...that has transformed the assay’s capabilities. Specifically this includes primer directed isothermal RNA...

  10. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ... many biological research situations, the relevant volume is that of the cell. For the physical behaviors...major applications of FPs is to visualize the localization, dynamics, and behavior of a POI. As an investigator...monomeric FPs are not actually monomeric, at least in practical terms. The propensity of a particular type of ...fusion protein, as in a FRET biosensor, then the local concentration of FPs around that fusion protein ...consequences can be dramatic. For a membrane POI localized to the endoplasmic reticulum, the normally spiderweb-like...structures are large (microns), bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to...fusion proteins that form as a result will often localize incorrectly and may function improperly. 3. Incorporation...

  11. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    Published
    Nov. 27, 2019, 2:03 p.m.
    ... the brain. A more locally defined set of neurons can then be targeted with optical stimuli. The restricted...viral vectors (AAV), neuroscientists can combine classical genetic tools with the manipulation of specific...of these discussions as a publicly accessible practical guide which was recently published in Frontiers...of molecular tools and sensors Combinations of classical genetic tools, for example Cre/loxP or Flp/FRT...transgene expression by restricting expression to genetically defined cell types. Specific transgene expression...result in opsin expression in their cell bodies. Optical stimulation in a different brain area can then ... more versatile. To complement the wealth of genetically encoded tools available, researchers are also...

  12. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ... In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols,... smaller pyrimidine:pyrimidine mismatches are typically worse ligation substrates than pyrimidine:purine...design and reaction condition optimization, will be critical for success. Online tools are available to help... the Thermostable Ligase Reaction Temperature Calculator can be used to estimate an appropriate incubation... structure, reaction mechanism, and function." Chemical reviews 106.2 (2006): 687-699. PubMed PMID: 16464020... ligases: progress and prospects." Journal of Biological Chemistry 284.26 (2009): 17365-17369. PubMed ...reaction-capillary electrophoresis assay." Journal of clinical microbiology 45.6 (2007): 1927-1935. PubMed PMID...

  13. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

    Type
    Blog Post
    Published
    Feb. 13, 2018, 2:51 p.m.
    ...interaction with limited biological materials. Problems with Chip-seq ChIP-seq typically requires a large amount...activity of a Protein A fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound by...material for a ChIP-seq experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking during...development using the Drosophila wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings...addition of Protein A MNase. Once MNase has been localized to target sites, the nuclease is briefly activated...targeted digestion is controlled by the addition of calcium, which MNase requires for its nuclease activity...and simplification of the original technique, specifically in terms of initial steps of cell or tissue ...

  14. Addgene-Seeding Labs Plasmid Grant: Accelerating Science Globally - 5 Scientists, 4 Countries & 3 Continents!

    Type
    Blog Post
    Published
    July 18, 2017, 12:41 p.m.
    ...screened for toxicity against local insect pests. By developing biological agents such as insecticidal ...get tested for their potential agricultural and medical use. Find More Optogenetic Tools Like pDawn  Making... improving expression of nutritionally and therapeutically significant proteins. In this project she aims...insecticidal proteins, the use of chemical pesticides may be avoided or decreased. Biosensors for herbicide detection...one of the reaction’s metabolites. One useful genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which...working in a wide range of research fields and geographical locations. We are excited to see how all of ...et al. “Endophytes as sources of antibiotic” Biochemical Pharmacology 2017 Jun 15;134:1-17. PubMed PMID...

  15. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    Published
    Dec. 7, 2021, 2:15 p.m.
    ...Immunoprecipitation Overview An immunoprecipitation reaction is typically carried out in one of two ways. Both methods utilize.... 2) An antibody immobilized on a bead binds specifically to the blue triangle protein but not the other... to the process of anchoring an antibody, often called the capture antibody, to agarose or magnetic beads... or magnetic beads with commercially available chemical agents. Directly conjugating the primary antibody...interactions To prepare for an IP, samples are typically lysed in a non-denaturing buffer containing non-ionic...sample to remove any proteins that may interact nonspecifically with the capture antibody or Protein A/G. To...interaction and/or the downstream application. Typically, if the target protein will be detected in a Western...

  16. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    Published
    April 2, 2019, 1:44 p.m.
    ... well as neural development. Specifically, SHH is a morphogen, a chemical signal that diffuses down a ...exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles) outline...on the spindle of a spinning wheel but also a biological transposon system. Sleeping Beauty is a synthetic... cellular contraction You may think that a gene called "spaghetti squash" originated from the squash itself...contract depending on the context. Contraction is critical for processes such as cell movement/motility, ...or dead cells (Jiang and White-Cooper, 2003). Why call a gene required for spermatogenesis after a Sesame...mesoderm, is necessary to activate bagpipe which is localized to segmental clusters of the mesoderm. This subdivision...

  17. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    Published
    July 15, 2025, 1:15 p.m.
    ...the nanopore, the electrical current recorded by the instrument changes. Base-calling software can then...then separated by size using electrophoresis, typically capillary electrophoresis. Capillary electrophoresis...sample at a time, which can become costly and impractical for whole-genome (and often, whole-plasmid) sequencing...many samples and can output more data, and it typically results in high accuracy for full plasmids. However...attached to the flow cell membrane and have an electrical current running through them. DNA or RNA is pulled... can manually revisit the assembly in a program called Geneious. Within Geneious, the raw FASTQ reads ...sequences submitted by depositors. A sample will automatically pass through our QC process if there is a 100%...

  18. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    Published
    Nov. 18, 2016, 3 p.m.
    ...order in which specific chemical inputs are received by a cell. These so-called recombinase-based state...light and chemicals respectively. Didem Goz Aytruk from Connie Cepko’s Lab at Harvard Medical School describes...although fluorescent proteins are incredibly useful, chemical fluorophores can still be used in a wide variety...variety of experiments. Read more to find chemical fluorophores for your next experiment. Nucleic acid based...report upon and/or engineer “temporally ordered biochemical events” such as those that occur during development...to pH changes without the need for ATP or other chemical fuel sources.” In her work, Dr. Polka made R-body...repression of different genes in a single cell, and localization of a fluorescent signal to your target gene....

  19. HA Frankenbody, a New Imaging Tool to Visualize Single Molecules and Nascent Peptides

    Type
    Blog Post
    Published
    Oct. 10, 2019, 12:41 p.m.
    ...Different antibodies bind different protein sequences called epitopes and scientists can use these to tag their...lab used an alternative strategy to develop a genetically encoded antibody based probe that detects the... epitope in diverse cellular environments. They called this probe the HA frankenbody (Zhao et al., 2019...targets, the scientists fused them to sequences called complementarity determining regions (CDRs, or loops...concept experiment the Stasevich lab observed colocalization of GFP tagged HA frankenbody with an mCherry...meaning that scientists can track the movement and localization of single HA-tagged molecules over time.  The... Inbar, D., Hochman, J., & Givol, D. (1972). Localization of antibody-combining sites within the variable...

  20. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...by fluorescence photoactivation localization microscopy." Biophysical journal 91.11 (2006): 4258-4272....Berlin, Shai, et al. "Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging...tag cellular proteins and investigate protein localization, interaction, and expression. However, one caveat...oligomerize, disrupting cellular function and localization, or leading to protein aggregation. Another ...important applications for PA-FPs involve enhancing optical live imaging of proteins, organelles, and cells...proteins, scientists can track single protein localization, turnover, and trafficking, as well as organelle...new imaging methods, including photoactivation-localization microscopy (PALM, also FPALM) (11). For PALM...

  21. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    Published
    Sept. 10, 2015, 2:30 p.m.
    ...run it? And, most importantly, what are the methodological and experimental limitations and considerations...numerous scientists at NIDA and some of the practicalities and considerations you may not find in the ...stimulate 2) Fabrication and implantation of the optical fiber into the region of interest 3) Behavioral...region and cell type of interest is obviously of critical importance to any optogenetic experiment. There...pioneered the development of optogenetics, has genetically manipulated a number of the opsin genes to alter...your opsin  The standard opsin delivery method is local injection of an adeno-associated viral (AAV) construct... or more mice and the injections must be near identical for all of these surgeries. Identify coordinates...

  22. 5 Great Apps for Lab Life

    Type
    Blog Post
    Published
    Sept. 22, 2016, 2:30 p.m.
    ...MSDS for a lab chemical of choice. Now, I can’t guarantee every reagent in your chemical closet is in this...tried to find an app that could help me with calculations. This is how I came across the Gibco Cell Culture...culture and include a counter, lab timer, dilution calculator, and a couple of other nice features. This app...sequence for your enzyme of interest. Lastly, the Tm calculator helps you set up an optimal PCR depending on ...enzyme sequence resource is universally useful. 4. Chemical Safety Data Sheets by ThatsMyStapler Safety always...experiments. Hence, I’m including this MSDS app. The Chemical Safety Data Sheets app has a search function that... if you get quizzed randomly about a hazardous chemical and can’t find your lab’s MSDS book, this is a...

  23. March for Science

    Type
    Blog Post
    Published
    April 21, 2017, 2:30 p.m.
    ...general (1). Science and politics? Science is an apolitical process for seeking knowledge. The process begins... again. No part of this process is inherently political so why is there a march in Washington, D.C., the...States government? Politics and science are intrinsically linked: governments exist in reality and science...as evidence of climate change, the safety of genetically modified organisms (GMOs), the absence of a link... it.  3) Linking scientists to one particular political party while alienating the other While scientists... yet research shows that thoughts on some non-political issues are split along party lines (18). Feeling...alternative. Inclusion of data informing policy specifically related to the following subjects: Climate ...

  24. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    Published
    Aug. 15, 2019, 12:30 p.m.
    ...Belarusian State University Series of Physiological, Biochemical and Molecular Biology Sciences. 9.1 (...genomic modification. This technique is frequently called allelic exchange. Here, a mutated allele introduced...: The initial plasmid integration step (the so-called “single crossover”) which results in a merodiploid...They hypothesized that genes that had evolved specifically for toxicity would allow stronger counter-selection...system is well-characterized and quite tight (Giacalone et al., 2006). The Chen lab demonstrated excellent...synthetic ribosome binding site (using the online calculator from the Salis group) (Espah Borujeni et al.,...from TP997). We validated these vectors, which we call “pTOX” in a broad group of Gram-negative Enterobacteriaceae...

  25. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    Published
    June 23, 2016, 2:30 p.m.
    .... Lajoie, Marc J., et al. "Genomically recoded organisms expand biological functions." Science 342.6156...phosphorylation events on the activity of a given protein? Classical methods for studying protein phosphorylation A...plasmid. This includes 4 copies of a tRNA that specifically recognizes amber stop codons (TGA) to encode...effects on cell growth. The C321.ΔA strain was genomically recoded to eliminate all 321 amber stop codons...strain, TAG is an ‘open’ codon, which can be specifically recognized by the Sep-tRNA. 3. An expression...specific phosphoserine residue at this position. Practical tips for your experiments To generate as much ...containing Phos-Tag, a small molecule that binds specifically to phosphate moieties and promotes a robust ...

  26. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    Published
    June 25, 2019, 12:54 p.m.
    ...methods including both chemical and physical methods in the lab. Chemicals like calcium phosphate and diethylaminoehtyl...competent through chemical manipulation in the lab. This is commonly done using calcium chloride which permeabilizes...also use electroporation, the application of an electrical charge to cells, to increase cell membrane permeability...contact. The donor bacterium contains a DNA sequence called the Fertility factor (F-factor). The F-factor is...easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection...cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell ...

  27. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...fluorescent genetically encoded voltage indicator Article contributed by Shreya Vedantam Genetically encoded... for its biotechnological applications including conversion of CO2 into water and electrical energy generation...can be reconstituted in vivo to form a protein identical in sequence to the unmodified enzyme. Each half...tested the base editors in vivo and achieved therapeutically relevant editing efficiencies in the mouse ... base editors at Addgene. Levy et al., Nature Biomedical Engineering, 2020. PubMed PMID: 31937940. Detection...single action potentials and further studies of optical neural circuit manipulations when combined with...fusion proteins separate, inactivating Cre. This is critical for reducing Cre toxicity in long-term experiments...

  28. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...ability to specifically recognize and bind proteins. They’re used in a number of research and clinical applications...Instead, we're going to focus on what I'm going to call "alternative affinity reagents" - everything from... of the antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in ...other (Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are... from a camelid antibody lacking a light chain, called a heavy chain antibody (Muyldermans S., 2013). ...Designed ankyrin repeat proteins, or DARPins, are genetically engineered small affinity proteins, around 14...proteins known as ankyrin repeat proteins. DARPins typically consist of 3-4 repeats, have high specificity ...

  29. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    Published
    June 27, 2019, 12:25 p.m.
    ...detect mRNA from 10 bacteria of clinical significance and four clinically relevant host biomarkers. The ...microbiome with sequencing is vast, the ability to genetically alter microbiome members is limited and many ...at Columbia University developed a method for genetically altering gut microbiome members in their native...native habitat (Ronda et al., 2019). They called their method Metagenomic Alteration of Gut microbiome by... analyze gut microbiota and host biomarkers In clinical and diagnostic labs, deep sequencing and qPCR ...large number of microbiome samples. To enable large-scale, longitudinal studies of the gut microbiome, James...The lab validated their platform on clinical stool samples by comparison to qPCR and demonstrated that it...

  30. All in a Twist: dsRNA

    Type
    Blog Post
    Published
    Feb. 2, 2023, 2:15 p.m.
    ...almost always epigenetically silenced, but dysregulation of these systems can lead to local dsRNA production... DNA – right? RNA performs almost all of its biological functions in our cells in the single strand form...machinery of the host. This pathway has been clinically exploited for antiviral purposes similar to that...sequence. Once a compatible sequence is found (typically RNA that the RNAi is designed against), RISC can...target mRNA. These RNA tools have been used in clinical therapy to target pathogenic proteins, as well...and insecticides, an advancement over previous chemical versions with toxicity to many organisms. ...transcription of their circularized DNA. These dsRNAs are typically contained within the mitochondria, barring a loss...
Showing: 121 - 150 of 846 results