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...you can't distinguish the different proteins from each other. To distinguish different target proteins ... analysis, you'll need to use fluorophores that attach to the target. When you design a flow experiment...experiment, you'll need to pick a fluorophore for each target and ensure the fluorophores, together, are able...cells with fluorophore-conjugated antibodies that attach to a specific target. By using a mix of different...respective colors of a few commonly used fluorophores. Each fluorophore has a defined emission maximum. Note...to as bleeding. When two fluorophores bleed into each other, it means that the cytometer could detect ...used in flow cytometry. Let's briefly go through each section of this article again. In the beginning,...

...fluorophores can undergo FRET when they are near each other. How do you label your biomolecule of interest... harder due to tens of thousands of biomolecules each containing multiple binding crevices that can nonspecifically...resulting in site-specific incorporation of the attached fluorophore. However, since tRNA suppression competes...can be enhanced by coupling two NTA molecules to each Cy dye, as well as by extending the length of the...acceptor fluorophore binding sites are adjacent to each other on the same protein and a negative control... 269-272. PubMed PMID: 9657724. 6. Kapanidis, Achillefs N., Yon W. Ebright, and Richard H. Ebright. "Site-specific...Saraheimo, Satu, et al. "Time-Resolved FRET-Based Approach for Antibody Detection–A New Serodiagnostic Concept...

...optogenetics experiment and what are the tools required for each one? Behavioral Optogenetics can be broken down ...experimentation I’ll discuss some of the key features of each of these steps. Today we’ll discuss selection and...membrane hyperpolarization but unlike HR, this is achieved through hydrogen ion efflux; evidence suggests...expression of your opsin is no longer required to achieve cell-type specific opsin expression. However, the...take as long as 6-8 weeks for sufficient opsin to reach the axon terminal. Again, this must be empirically...neuronal populations using combinatorial viral approaches. Current protocols in neuroscience / editorial...PMC3191888. 9. Zeng H, Madisen L. Mouse transgenic approaches in optogenetics. Progress in brain research. ...

...approximately the length of your average PhD to achieve!) Each field has its own framework: a particular way...audience - like the professor who once decided that to teach high school students PCR, they first had to understand...but the following tips can help:  If there’s a teacher, mentor, or other educator working with your audience...working out, adjust the ask.  I used to do a lot of outreach to student groups and being aware of cognitive... Rachel Leeson...

...existing antibodies. Each cleaved antibody will produce two ~50kD Fab fragments, one for each arm of the antibody...bispecific, meaning that each scFv can target a different antigen; binding of each can be either simultaneous...antibodies can allow you to be flexible in your approach to affinity reagent applications. Whatever affinity...  Rachel Leeson...

...previously developed small molecule degraders that achieve selective degradation of endogenous proteins (notably...Hippel-Lindau (VHL) to bring the endogenous degradation machinery into close proximity with the target protein, ...to co-opt the endogenous ubiquitin proteasome machinery to degrade any protein. We are particularly excited...that are not possible with traditional genetic approaches. In addition, the dTAG system is versatile and...FKBP12F36V-fusion and the ubiquitin proteasome machinery through binding with cereblon, leading to rapid...Confirm the functionality of your fusion chimeras For each protein-of-interest, it is important to determine...

...depending on the conference you’re attending and who approaches you. You might also have cross-team projects ...picture of virtually every container we used, then for each listed: An “official” name for the container to...pretty Addgene specific) The types or purposes of each given container, when applicable Any other relevant...enough. We also had to enforce and maintain it. Teaching people to use words in a new way is not necessarily...Words are important!”  If you try any of these approaches in your lab or workplace, let me know. If you...you have come upon any other effective approaches for clearing up communications lines in your professional...

...looking for outreach volunteers as well. 4) Blog - Hone your writing skills Take the DIY approach and start...engagement - Participate in science festivals and outreach If you are comfortable talking in public, volunteer...Volunteer – Step into science education Do you aim to reach young people? Try networking with your local education... a blog is not only a great form of scientific outreach, it also helps you to hone your writing skills...Center for Communicating Science. If you choose to teach yourself, check out MIT’s OpenCourseware for course...

...Picogreen quantification on each and every isolated plasmid sample and normalize each to fall within a 10 fold...make use of a pipeline that takes the raw data from each sample in our pool and individually assembles the...plasmid sequences). As with all things at Addgene, each part of this new process involves a significant ...Confirming tags and fusion proteins. We’ll break down each of these steps below. Aligning the sequence The ... making sure to note the appropriate barcode for each sample. About 24 hours later we unload the FASTQ...

...to be less expensive than an ELISA kit. In this approach, viral RNA is first converted to cDNA and then...several different lentiviruses in parallel, this approach should be considered. Similar to the p24 assay... Fluoresence Titering Protocol Another popular approach utilizes the antibiotic resistance genes carried...have integrated into a target cell’s genome. This approach can discriminate multiple integration events, ...underestimating the viral titer. The main drawback of this approach is that it can be quite labor intensive. Target...virus may want to choose more straightforward approaches such as the FACS-based or colony-forming unit...

...design hundreds to thousands of different pegRNAs for each desired edit, it simply becomes impossible (or at... efficiency and building prediction tools with machine learning (PRIDICT, PRIDICT2.0).   How to apply...pegRNA efficiencies with pridict.it. This approach leverages the strengths of PRIDICT/PRIDICT2.0 ...chromatin-based prediction tool (ePRIDICT). In summary, machine learning-based prime editing prediction models ...computational biology. His expertise includes applying machine learning techniques to make sense of biology, with...B. van, Krauthammer, M., & Schwank, G. (2024). Machine learning prediction of prime editing efficiency...

...protocols attached.  Final step: validation  Remember, there is no “one-size-fits-all” protocol! Each library... we have an amplification protocol developed for each individual pooled library; generalized amplification...decide what transformation efficiency you need to achieve. Most pooled library protocols aim for somewhere...weight of your library plasmids. As long as you achieve sufficient coverage from your transformation, any...varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony, and...

...for in vivo delivery of advanced genome editing machinery such as CRISPR-Cas. Figure 1: Schematic...transduced to target cells. Within target cells, Cas9 machinery is transiently expressed and binds to target DNA...rates of targeted mutagenesis similar to those achieved by isogenic AdVs encoding TALENs targeting the...18% to 65% (Maggio et al., 2014). An alternative approach to NHEJ is to utilize the Homology Directed Repair... Adv vector delivery is often used, the CRISPR approach based on HDR has the advantage of being a potentially...). If sufficient gene transfer and knock-in is achieved, producer cells could produce corrective amounts...

...is to make it detectable. Visualization can be achieved using fluorescent microscopy - even in living ...membrane permeabilized in order for the antibodies to reach their intracellular targets. Therefore, it can be...setup for a different fluorescent signal) and photobleaching i.e. FP stability. One should also be aware...make sure that the selected FPs do not influence each other and thus potentially falsify the signal. As...Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04... Susanna Bachle...

...-colored protein from the jellyfish Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years later...allows photosynthetically active wavelengths to reach zooxanthellae, the coral’s symbiotic dinoflagellates...Chromoproteins also enhance corals’ resistance to mass bleaching, the loss of their symbiotic algae, during heat...cost and resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment...agar plate. Image: Liljeruhm et al., 2018. For each chromoprotein, the lab determined the color intensities...protein from the mangrove jellyfish Cassiopea xamachana. Marine Biology 94:423–430 . https://doi.org/10.1007...

...cells, attacking the cellular machinery necessary for division. This approach also kills dividing non-cancerous... of people die from cancer-related complications each year. Despite these grim statistics, new strategies... University of California, San Francisco, is approaching the Ras problem by exploiting the different mutant...the generation of mutant cell lines. Further, for each plasmid in the kit, the transcript most commonly... collection of cancer-related plasmids such that each is linked to a known major pathway. Now, using our...

... more to achieve maximum plasmid yield. The optimal growth time should be determined for each plasmid/...number, the number of plasmid copies supported within each bacterial cell in your culture. Copy number is partially...amount of DNA you purify! Additionally, if you've reached your lysate volume limits, the copy number of some...concentration. Add supplements to the media You can achieve higher cell density using a nutrient-rich broth...insufficient gas exchange will prevent your cultures from reaching the desired density. The volume of your flask ...

.... More and more CRISPR tools are being published each month, making CRISPR a great choice for your next...expression systems, the typical applications for each, and potential delivery methods.   General considerations...Determine what type of outcome you are trying to achieve: Do you want to permanently knock out or knock-... All of these outcomes can be most effectively achieved with different CRISPR components. Select the appropriate...target cells.  Mammalian CRISPR expression systems Each model system has its own best practices for efficient... summarizes the various components included with each expression system as well as suitable applications...

...composed of many different gRNA-containing plasmids, each with a different genomic target. The goal of a CRISPR... of gRNA-containing lentiviral transfer vectors, each targeting a specific gene within the genome (see...a heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a ... plasmids, discerned only by a unique barcode on each plasmid. As such, sequencing CRISPR libraries after...target gene, and maintaining the distribution of each gRNA within the population is key. Loss of representation...gene is actually important for a given phenotype. Each hit from the screen should be independently validated...

...while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination pathway...protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification kits..., but have successfully tested DH5a, TOP10, and Mach1. No common lab cloning strain has failed so far....complex assembly of multiple genes. Primer design for each type of modification is shown in Figure 2, and is...site. Encode half the new sequence at the 5’ end of each primer, with an overlap for homologous recombination...in a gene, while adding a GFP-tag (Figure 3). Achieving multiple modifications requires multiple recombination...