.... 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...

...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:...

..., 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...

... 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...

...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...

...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...

...these types of bigger picture problems systematically and logically is as useful for finishing your thesis... but this skill is not just about reacting to technical issues. This skill also comes into play while ...defining problem solving skills as: One’s ability to logically think through a problem/task/situation, come up...Problem-solving overlaps heavily with other skills such as critical thinking, decision making, creativity, or data... really are, while also practicing systematic, logical thinking.  If you are already a lab veteran at ... to troubleshoot; you probably did not just automatically come to a solution. Consider the thoughts that...solutions. Ultimately, problem solving is less about technical know-how and more about how you approach new challenges...

...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 ...sequences for your next CRISPR experiment.    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...

...Stanford University. Sex and gender are critical components of biological research that are often forgotten ...the past decade. For more practical help, there are numerous methodological articles to consider—we have... that of human clinical trials. And, as Janine Clayton points out, late-stage clinical studies that draw...effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to why... disease being treated, and other hormonal, immunological, and environmental factors. How to include sex... (NIH) now requires that sex be included as a biological variable in all NIH-funded research. Janine Clayton...

...generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem cell reprogramming...clones. Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors...lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox lentiviral vectors...diluted faster when cells are actively dividing. The typical drawbacks of using transfection will still exist...DNA-silencing mechanisms. While minicircles are typically smaller than traditional plasmids, Jia et al generated...determine how stem cell-like they behave. iPSCs are typically tested with molecular-based assays first. These...molecular hallmarks of reprogramming, then they are typically assessed with more rigorous functional tests. ...

...regular part of basic biological research as well as clinical therapy. These biological tools are useful because...  When viruses were first being engineered as biological tools, thought was given on how to keep the replication...encoded in the transfer plasmid, the component that physically integrates into the host genome, the virus is...caution as they are integrating viruses – they physically deposit their cargo into the host’s DNA. These...should be thoroughly decontaminated after use, typically with bleach (plastic) or ethanol (other surfaces...Virus Many virus experiments involve needles or scalpels (sharps). Cuts and scrapes from sharp equipment...specific organization/department/lab’s policies – basically, you’ll need to get trained in each new place ...

...relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference controls...spectral flow experiment and panel, there are a few critical factors to keep in mind in order to get the best...extraction Cells produce a low level of fluorescence called autofluorescence (you can see this in your unstained...controls). Spectral flow cytometers include a tool called “autofluorescence extraction” which allows the ...autofluorescence across various tissue types varies, it is critical to include an unstained sample for each tissue...detected. Cytek’s website has a convenient tool, called the similarity index, which allows the user to ...and a value of “1” indicating those markers are identical and completely indistinguishable. This tool is...