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...tricks By Ashley Waldron While IHC protocols typically call for thin sections of tissue, the Wu Lab is ...therapy, Feng Zhang’s lab has developed a system called DIRECTED (Delivery to Intended REcipient Cells ...promising approach for experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger...labeling can identify protein interactions or map a local proteome, but some applications have been challenging...control over when and where the enzyme is active, drastically reducing background labeling caused by the biotin...proximity labeling enzymes, TurboID and APEX2, are localized to different cellular compartments. They showed...of a cell. Lin et al. generated a fusion protein called iPAK4 that assembles into a slow-growing protein...

... general public. Chemical Society in University College Dublin, Ireland The Chemical Society was founded...way up to professors. Oxford Biological Society, England The Biological Society based at the University...idea of the Freshers Fair and some of the great biological societies that Addgene has had the opportunity...at Freshers Fairs in the past year. Cambridge Biological Society, England Since 1996, Cambridge Biosoc...Biosoc’s goal has been to promote interest in the biological sciences amongst the student community at the...At Addgene, we’re incredibly glad to see these biological societies engaging with young scholars. We believe... to keep up the good work! What scientific or biological societies were you a part of in undergrad or ...

...graduate students and postdocs to think analytically and critically. Passion fuels the drive to continue ... Connie Cepko (Harvard Medical School) , and Dr. George Church (Harvard Medical School) for taking the...people (especially you!) care about. Have you critically evaluated your plan? Have you read the relevant...write a grant for this project, what would be your critical experiments and why? What techniques and skills...help move your project forward? Most projects typically require a diverse set of skills. It is important...direction of the project? What is your timeline? A typical research project can take several years to complete...time to generate new ideas and think about them critically before diving in. Research projects are designed...

...transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for many fields of biology and enable the...Nanobodies are single domain antibody fragments typically engineered from heavy-chain only antibodies found... with specific genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered...isolated and analysed. In this work, the authors specifically investigated protein binding at the TERT gene...his group from the University of Massachusetts Medical School have developed a group of plasmids (here...only 1 min. Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed...by mass spectrometry allowing a researcher to specifically identify proteins that are enriched in the 20...

.... Factor Xa loves calcium, but metal-chelate matrices, not so much. Maybe a calcium-independent proteinase...This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting ...subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized...derivatives streptavidin and neutravidin) is for all practical purposes covalent. Therefore, protein immobilization... four biotin-binding sites per molecule so, theoretically, saturated avidin could form tetramers of biotinylated...well-controlled. The lack of site selectivity typically diminishes the apparent affinity for the ligand...However, large amounts of the target protein are typically required due to the relatively low efficiency ...

...The good news is that viruses are very valuable biological tools, and with the right safety considerations...infiltration into a cell. The twist is that the typical genetic cargo — the viral genome — is swapped, ...vector containing the cargo of interest is then typically transfected along with viral packaging plasmids...risk management when working with viral vectors. Typically, one day after transfection into the production...expression or integration resulting from transduction typically plateaus within a few days to a few weeks, depending...context you are targeting? Second, what is the biological outcome you want to achieve? For example, do ...swap out your virus type to achieve different biological outcomes. For example, some viruses integrate...

...humanistic and philosophical implications. As an example, a group at Caltech used an optical fiber together...Optogenetics is a toolset that lets you turn the electrical activity of neurons on or off using pulses of...photosynthetic or photosensory molecules that come from basically every branch on the tree of life. Our most recent...some of these mysteries. Another application is clinical; can we find ways to treat patients? Over a quarter...implant already. If we can use light to more specifically activate a set of neurons, maybe we could treat...modulate electricity. If you could selectively let in calcium or some other specific ion that would be interesting...Addgene Alerts box. References: Independent Optical Excitation of Distinct Neural Populations. Nathan...

...situation in cells. Therefore, the localization of critical fusions (e.g. tubulin or connexin) has been...needed for Golgi localization, is occluded and therefore this protein is mislocalized. The APT1-mVenus... from Aequorea victoria and its variants are genetically encoded fluorescent probes. One of the limitations...ultracentrifugation or gel filtration is the most common analytical method. However, these may not reflect the situation... the POI. For example, the APT1-mVenus fusion localizes at the Golgi, whereas the mVenus-APT1 fusion does...function of the native protein that is tagged. The localization of the fusion protein can be verified and it...fluorescent protein fusion may never be able to act identical to the untagged protein, the benefits of being...

..., you’ll first need to learn a little bit of theoretical background on the different parameters that your...and W describe the shape of a histogram, which graphically represents the time and intensity of the cell...Live/Dead gate to focus your analysis on what’s biologically active. After that, the next gates fully depend...depend on your individual experiment. Practical example: lymphocytes in a mouse tumor sample You made ...made it! So much about the theoretical background. Let’s dive into the praxis of gating strategies. As an... would work just as fine (Figure 4). As H and A scale in proportion (while H and W do not; see Figure ...presented with a trend of two populations with basically a smear in between. Again, it can depend on the...

...hybridoma monoclonals, we anticipate that our chemically identical hybridoma-derived antibodies will also work...specific ligand-binding functions, all key to basic biomedical research and drug development. Integrin structure...long legs that protrude from the cell membrane, physically connecting the extracellular environment and ...signal cascades, and allowing agile responses to mechanical forces in the cell’s microenvironment. The actin...and (C) extended-open. When a ligand binds, it typically shifts the integrin from its closed to its open...resulting in cells that elongated and morphed asymmetrically (Jo et al., 2022). By contrast, LVDP-binding...conferred less force, leading cells to spread into symmetrical circles. While elucidating the role of tension...

...or macromolecule. Biotin can be added enzymatically or chemically. Enzymatic biotinylation occurs naturally...Avidin is a glycoprotein that consists of four identical subunits that can each bind one molecule of biotin... Scientists have developed several methods to chemically biotinylate macromolecules of interest both in...acid functional groups in vivo via specialized chemicals (biotinylation reagents). There are a variety ...the specificity of the E.coli BPL, BirA. If you recall, BirA biotinylates only one lysine in the entire...using a biotinylated primer. Biotin can also be enzymatically incorporated during PCR using a biotin labelled...modification of exceptional specificity.” Trends in Biochemical Sciences, 24.9 (1999b): 359–363. PubMed PMID:...

... Biosensor AAV (calcium sensors and GABA sensors) Calcium sensors The latest calcium sensors from Janelia...include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases...for monitoring neuronal activity in the form of calcium dynamics. (For more details on this technology,...technology, see our blog post Overview of AAV Encoded Calcium Sensors). We are expanding the inventory of these... publication (Chettih & Harvey, 2019), “this localization should improve the specificity of influence ...Recombinase AAV Targeting and manipulating specific, genetically-defined neuronal populations with intersectional...pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5) Calcium Sensors pGP-AAV-syn-jGCaMP7b-WPRE (104489-AAVrg...

...protein based research. This year, we put out another call for applications. We had an overwhelming response... variants one at a time is time consuming and unscalable.  Melissa Chiasson, a graduate student in Douglas...of sequence variants on abundance simultaneously called VAMP-seq, for Variant Abundance by Massively Parallel...epoxide reductase (VKOR) variants. VKOR is an enzyme critical for activating clotting factors and is the target...instrumental in interpreting human variation at large scales and making personalized medicine a reality,” Chiasson... Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin. According...replicate current knowledge, but the sensor can also kinetically outperform current methods. Unger plans to use...

...To our surprise, we measured identical dissociation rates for catalytically inactive dCas9 molecules. We...these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and...(Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision...key scientific principle: that understanding a biological process in detail often suggests new strategies... dissociation rates and discovered that the catalytically active form of the nuclease dissociates very...Our in vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure...this publication were performed using a technique called nucleofection, which introduces functional Cas9...

...tools from across the biological spectrum to learn more about the brain and neurological disease. I was surprised...and, like many biological fields the number potential of experiments enabled by physical, software, and...brain functions and vice versa. Even within the biological sciences, there’s plenty of interaction between... microbial DNA they all seem to require some mathematical know-how for analysis. If you’re looking to ...presentation area, I honed in on projects using biological tools. In addition to the researcher using sequencing...but are afraid you don’t have the right kind of biological expertise to get into the field, there’s likely...genes to many different cell types with minimal immunological effects. AAVs are used in experiments targeting...

... finding new factors in your biological system of interest, the so-called “fishing expedition.” Your first...population (typically cells or small organisms) and then assaying for a functional or phenotypical outcome...that both attempt to identify new players in a biological process. Players can be proteins, genes, enzymes...obligatory growth advantage – that is, you are not specifically looking at cells that can survive. Instead, ..., the entirety of the small molecule realm is typically not feasible to test in any given screening experiment...selection process. Non-functional traits will be automatically eliminated, unlike in screens. Selections begin...the answer that much faster? In reality, many biological questions are not conducive to selections and...

...experimental RNA. To solve this problem, gRNAs can be chemically modified to resist degradation while still effectively...purchasing your gRNA. Pro tip: modifications are typically incorporated at the termini of the crRNA molecule...oligonucleotides. Some of these synthetic modifications, specifically ones which do not contain a 5’ triphosphate ...or immunity itself is being studied.   Chemical RNA modifications to improve stability.   ...targeted editing events. Both technologies utilize chemical modifications to the RNA to control the entire...visualize transfection efficiency and even track localization in cellular experiments. There are many suitable...References Hendel, A., Bak, R., Clark, J. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome...

...with biological protocols and revolutionary physical approaches. However, with each new technical achievement...aim to make microscopy as a tool accessible to practically everyone.     Fig. 1: Open source hardware...modify and optimize the code, find and fix security-critical bugs, and contribute to constant updates of the... OpenFlexure microscope or open-source modular optical toolbox openUC2 are trying to overcome this hurdle...reproduction of the structure in a similar way to the biological protocol, and assembly designs based around the...towards more open research. The EU, for example, is calling for the opening of sources within the next few ...relying on tailored image processing and low-cost optical setups. References Joel T. Collins, Joe Knapper...

...which let the team detect its co-localization with a membrane-localized mCherry or GFP protein. With this...it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to the...The opening and closing of the AsLOV2 domain allosterically alters the shape and binding ability of its...unmodified proteins. Affinity purification methods typically require a protein to be tagged, which limits these...provides greater spatiotemporal resolution than pharmacological or genetic manipulation of the signaling pathway...and differentiation.  Since Ras is a membrane localized GTPase, the team fused a mCherry OptoNB to the...fusion protein in cells that expressed membrane-localized mCherry. With darkness, SOS binds Ras and initiates...

...have such epitopes. These classical 'undruggable' targets are often intrinsically disordered and structurally...suppressors to growth factors, some of the most clinically-relevant proteins are also the hardest to study... detection of proteins without the challenges typically occurring with a fluorescence-based approach. ...species that can lead to phototoxicity, perturb biological processes, and compromise cell viability (Laissue...complementary validation of protein expression and localization. HiBiT-tagged targets in translational research...ubiquitin ligases in the degradation of CFTR, specifically UBE3C. Degradation of CFTR was measured through...luminescence signal, associated with endosomal localization of EGFR. This method offered kinetic, non-invasive...