...accurately calculate the protein concentration of any experimental wells in that range of optical densities.... When an ELISA kit is ordered, the wells are typically already coated with the antibody. Then, you add...process is the addition of a detection antibody (typically a monoclonal antibody) that also recognizes the... of enzyme present in the well and is used to calculate the amount of your protein of interest that is...is present. Using a Standard Curve To do that calculation, you’ll need to include a standard curve in your...concentrations of your protein of interest. The optical density (OD) - the measure of light absorbed by...samples can then be plotted along this line to calculate the protein concentration. Image from Jagarlamudi...

...et. al.). Physical versus functional titer Viral titers usually come in two flavors, physical or functional...the target cell’s genome. Physical titer The two most common assays for physical titer are direct p24 measurements...functional. Physical titers measure the amount of viral particles in a sample and are usually based on the...produced can actually infect cells. These assays typically involve infecting a target cell line with your...methods tend to be more accurate than measuring physical titer, there is a risk that they may underestimate...target gene. Developing the proper control line typically requires transducing with a limiting multiplicity...will the target cell type used. Therefore, it is critical that standard conditions be used when comparing...

...protein expression for experimental, biotechnological, and medical applications. Researches can visualize...of proteins including antibodies and pharmaceuticals, specifically interleukins. Yeast expression systems...tagging them with fluorescent proteins to study localization or purify proteins to study their structure,...these types of insect expression systems can be scaled up for production of large amounts of protein. ...time consuming as plant tissues themselves are biochemically complex. To circumvent these issues, scientists... taken advantage of the natural secretion of biochemicals and proteins through plant roots. Tagging recombinant...easily adapted to fermentation allowing for large-scale, stable production of proteins. In general yeast...

... curve to calculate its experimental value. Though it varies from assay to assay, typically the experimental...before running a western blot. Standard curves are critical for a number of popular scientific applications...curve by serially diluting a known control sample, called a standard. The ideal standard curve has at least...enzymatic assays are sigmoidal. Once the trendline is calculated, you can extrapolate the concentration of the...measurements and the trendline y = 0.6561x + 0.019 calculated. The concentration of an unknown sample is determined...standard curve scientists use graphing software to calculate the data’s coefficient of determination, R2. R2...running assays that rely on a standard curve, it is critical that the unknown sample’s concentration lies within...

...COVID-19 cases each week. Statistical Modeling Note -- If statistical modeling isn’t up your alley,... of Chemistry and Chemical Biology at Harvard University; and Howard Hughes Medical Institute Investigator...as expected, plasmid orders (left, blue) drop dramatically when there are many new COVID-19 cases being...order several plasmids at once, breaking the statistical independence assumption of the Poisson process...our distribution, resulting in the following hierarchical model: Because the U.S., Europe, and China ...multivariate Gauss-Hermite quadrature, since the analytical expression for the data likelihood contains an...His research uses applied machine learning and statistical methods for fundamental scientific discovery ...

... 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...

...of Various Motifs is Critical for Initiation of Replication. Journal of Biological Chemistry, 273(14), ... replication machinery. For instance, the ori typically contains iterons that serve as recognition sites...Acinetobacter spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Aliivibrio salmonicida, Anabaena spp., ...Gram-negative and Gram-positive  Acinetobacter calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces...Aeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium...pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens, A. salmonicida, Agrobacterium...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E...

...connections to that population. Limitations of common genetically encoded neuronal tracers Neuronal tracers such...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live...tolerated by cells. They can be used for protein localization, isolation, and tracking experiments (Rizzo,.... Thus they have created antigenic protein tags called “spaghetti monster” fluorescent proteins (smFPs.... Indeed, to create the smFPs Looger’s team strategically inserted 10 to 15 copies of single epitope tags...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...postsynaptic cell adhesion protein that plays a critical role in neural development. It belongs to a huge...

...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...

...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...

...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 ...

...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...

..., 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...

...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:...

... 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...