...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean abandoning...graduate training. PhDs have many roles to play inside, on the outskirts, and entirely outside of academia...

...chemicals, and are not always consistent. Precast gels, while expensive, reduce protocol time, can be ...chemical-based blockers, while much more expensive, are consistent and stable at room temperature for 1–2... Direct detection method Less sensitive; usually more expensive Nitrocellulose or low-fluorescence...Multiplex Fluorescent proteins Less sensitive; more expensive Strip/Reprobe PVDF membrane Increased...specificity, lower cross-reactivity, and are more consistent lot-to-lot. The highest reproducibility comes...quantitative assays. On the other hand, they are not as sensitive to lower amounts of protein, and can require more...  Lysis buffer When choosing your lysis buffer, consider the subcellular location of your protein. Proteins...

...antibody is less sensitive than HRP RANbody detection, it is approximately as sensitive as detecting the...detection is less sensitive than HRP RANbody detection but approximately as sensitive as antigen detection...approximately as sensitive as detecting antigen with a standard primary/labeled secondary Less sensitive than HRP-RANbody...approximately as sensitive as detecting antigen with standard primary/labeled secondary   Less sensitive than HRP-RANbody...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to...secondary antibody. Chicken IgY This RANbody reporter consists of two chicken IgY domains. Chicken IgY domains...detected enzymatically with HRP substrate More sensitive than detecting antigen with a primary/ labeled...

...detailed below. Voltage Sensitive Domain (VSD) GEVIs Simply put, VSD-based sensors consist of a VSD fused to...gated ion channels all depend on transmitting and sensing electrical potential. Thankfully, there is a tool... These tools have two main elements: a voltage-sensing component and a fluorescent output. The GEVI family...divided up structurally into 2 main groups: voltage sensitive domains and Rhodopsin containing GEVIs. Chemigenetic...fluorescent reporter and the mechanism of their voltage sensing component, which separates them into two distinct...indicator, which can make the kinetics of voltage sensing to signal output slightly slower than other approaches...approaches. Functionally, the voltage sensitivity of the available VSDs has been successful even at the...

...      Figure 1: A protein gradient. The density of the bands, which increase as protein concentration... buffers and strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing...re seeing protein aggregation, you may want to consider changing your incubation temperature. Some proteins...aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, ...If you need very accurate protein measurements, consider using an unstained protein ladder, which will ...ladder on the lefthand side. Whatever you do, be consistent and document it.   Did it work? Save yourself...reversible staining method of choice is appropriately sensitive. If using a PVDF membrane, you can dry the membrane...

...Specific ​H​igh ​s​ensitivity ​E​nzymatic ​R​eporter un​LOCK​ing) technique Sensitivity: Single molecule...Two-step-SHERLOCK has a sensitivity in the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar...for rapid, ultrasensitive and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies...SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-100 copies of RNA per microliter Time: ~40...two crRNAs that bind to two regions improves sensitivity of the assay. This reaction also contains SSB...one-temperature reaction, and is nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection...detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of RNA per microliter Time: Not mentioned...

...the loading control: the intensity of each POI band is divided by the intensity of the loading control ...band intensity measurements become extremely susceptible to error, since tiny changes in intensity can ...membrane, which would be directly reported by band intensity. Researchers use later steps in this chain (blue...trust Assumption #1, any change in the normalized intensity of your POI could reflect either a genuine change...relationship between protein abundance and band intensity is an S-shaped curve. Sparse proteins may not ...problems for proteins of very different abundance, consider the following example gel. Figure 5: An... on the plot of protein abundance versus band intensity (right). Created with BioRender.com. On this...

...conditions. The use of low intensity light solves each of these problems; inexpensive tools such as LEDs can...precise, consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength... low-intensity light, so off-target effects should be limited. Phytochromes are light-responsive systems...Tabor have been working on making E. coli light responsive since 2005, when Tabor was working in Voigt's... heading of two-component systems (TCSs). TCSs consist of a histidine kinase that phosphorylates a response...Christopher Voigt’s lab created the first E. coli light-sensitive two-component system (TCS). Levskaya et al. fused...Tabor et al. subsequently engineered a second photosensitive TCS activated by green light; these two systems...

...published Aug 30, 2018 and updated April 16, 2020. Sensitive and specific nucleic acid detection is crucial...nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at...at low concentrations and/or are too expensive, time-consuming, and complex to use outside of standard...and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at...and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2...CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (C2c2) was... your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or RNA from a sample...

...are not as sensitive, however, so those studying low abundance antigens may want to consider alternative... ELISAs for their accuracy, specificity, and sensitivity (a good ELISA can detect picogram quantities ...advantages and disadvantages of each, and some considerations when using the method. ELISA basics All ELISAs...detectable product. Scientists compare the signal intensity of the sample to that of a standard curve to extrapolate... themselves, the process can be laborious and expensive. In some cases, conjugation methods can interfere...Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary antibodies...sandwich ELISAs, in a competitive ELISA the signal intensity is inversely proportional to the amount of antigen...

...retain fluorescent intensity during exposure to light, is another factor to consider. If your application...interiors to many inquisitive scientists. Seeing inside these specimens has offered illumination on biochemical...will outline some fundamental characteristics to consider when selecting a dye or ligand to make your selection...proteins would be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme variant tag that...is bound through green light emission. Key considerations in selecting fluorescent dyes/ligands Effects...-conjugation affects protein function and its propensity to bind cellular components. Some dyes target...stably combined with nanoparticles, maintaining intensity at ~85% of its original value after 60 minutes...

...subcloning, expansion, and confirmation. This procedure can take several months but should be considered in labs...benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here...results in less than a day, but tends to be less expensive than an ELISA kit. In this approach, viral RNA...lentiviruses in parallel, this approach should be considered. Similar to the p24 assay, titering via measurement...When using qPCR based methods for viral titration consider using a reverse transcriptase mutant in a separate...carryover. Functional titer Functional titer is considered a more accurate method to quantify virus as it...of this approach is that it can be quite labor intensive. Target cells are transduced with serial dilutions...

...the least expensive of all the options discussed as it only requires PCR and the inexpensive DpnI enzyme...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...the following cloning methodologies have been extensively described in other articles; so if you are interested...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...

...cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience...particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes... calcium-binding sites as possible for added sensitivity. As reported in Nature Methods, the FRET changes...essentially all of the standard organisms for which extensive genetic tools are available. While he recognizes... he said. “It gives you more information than intensity-based readouts.” Griesbeck also points out that...approach he and his colleagues took to optimize sensitivities can now be applied to dozens of other published...for visualizing the transcription factor cAMP-responsive element-binding protein (CREB) in living cells...

... One consists of NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc... (FRET) probes. These probes generally consist of a responsive protein (e.g. calcium binding protein) ...membrane-inserted voltage sensing domain (LOTUS V; Luminescent Optical Tool for Universal Sensing of Voltage). With...how bright you can make a reaction but for how sensitive you can make a reaction. NLuc requires 100-fold...) fused to two fluorescent proteins. When the responsive protein binds to calcium it changes conformation.... In well-designed FRET-based biosensors, the intensity of the FRET signal can be correlated to specific...No photoexcitation High Ion specificity High sensitivity Easy expression Gives ratiometric data The result...

...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...different opsin (natural and engineered proteins responsive to light in combination with retinal) variations...reviewed in Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS (OptoXRs) (Tichy et al., 2019),... the extracellular domain of endogenous light-sensitive receptors, like mammalian rhodopsin, and the intracellular...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...

...your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from your...Addgene uses and recommends DH5𝝰! This strain consistently gives good, high quality preps due to its endA1... this handy guide from OpenWetWare for a more extensive listing of common E. coli strains. Increase the...media or adding supplements can improve your cell density, leading to an improved plasmid DNA yield. Culture...supplements to the media You can achieve higher cell density using a nutrient-rich broth such as Terrific Broth...in moderation) may also serve to increase cell density and plasmid yield. Start with a single, fresh colony...Increasing the incubation time can improve the cell density and therefore the plasmid yield. You can optimize...

...each event to a channel number based on intensity. Higher intensity events are assigned to higher channels...a user will compare the shifting intensity of events. Since intensity positively correlates with expression...cabinet with cloning rings or following labor-intensive limiting dilution protocols? If this sounds like... the cell’s size, complexity and fluorescence intensity.   What flow cytometers detect Cell size and... a high degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression...The electrical system converts the fluorescence intensity to a voltage pulse, called an event, and assigns...reporter on the N- or C-terminus of the protein. One downside of fusion proteins is that the tag can alter the...

... to be considered as well. In this post, we’ll touch on antibody storage, buffer considerations, and give...records, and consider the following questions as you move forward:  Was a light-sensitive conjugate on...you’re planning to run with the antibodies time-sensitive, using precious samples, or being compared to ...data in a manner that demands high accuracy and consistency?  If the answer to any of these questions is...saving the poor, abandoned antibody for a…less sensitive moment.   Fig 1: Blugene fading into darkness...fix it. HRP is easy to find, simple to use, not sensitive to light in its inactive state, and cheap. Unless... panel in the machine. Fluorophores are light sensitive, so antibodies conjugated to them need to be stored...

...more sensitive channelrhodopsin actuator for triggering action potentials at low light intensities, the...cumbersome. To overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or...antibodies fused to sfGFP. This system amplifies the intensity of fluorescence signal and enables tracking of... H+ specific response cytoplasmic retention insensitivity to other ions In BRET, A mutant Renilla luciferase...dimers by fusing the SsrA peptide with the light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2...activator protein to drive transcription. The anchor consists of dCas9 fused to CIB1 and is targeted to specific... a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm) to far-red (λ...