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...proteins within a system. This can be pretty complicated given that a single cell has tens of thousands...single protein? Oftentimes, they rely on protein purification. In this strategy a single protein can be isolated...this blog will focus on one of the most common, purification based on the polyhistidine tag. What is the ...purity (Janknecht 1991, Hochuli 1988). In this purification approach, the metal ions are immobilized on ... incorporation of a cleavage site and a post-purification cleavage step to remove the tag in order to ...2000).  One downside of polyhistidine tag-based purification, however, is the propensity for nonspecific ... there is no cut or dried answer. Successful purification requires the tag to be accessible to the metal...

...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important ...normally one figure and can be negative and/or even replicated studies to show reproducible results. This a ...referenced. In addition the data published in microPublications is curated and added to other databases including...including wormbase and flybase. microPublication is run and maintained by members of the worm community...introducing point mutations that was published in a microPublication (Schwartz and Jorgensen, 2018).  These are...

...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...and incubation times. If the vendor includes publications that cited the antibody, consider reviewing ... the materials and methods sections of those publications.  Finally, always verify that the chosen antibody...

... its nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being...characterizing the 4D nucleome. Future modifications and applications Ma et al. developed this technique ...have major applications to cancer, for example, the visualization of chromosomal translocations or chromosomal...plasmids from this publication at Addgene. A protein-tagging system for signal amplification in gene expression...technique published every week! One of the newest applications is a colorful system that uses fluorescently...Feb 23. Pubmed. Find the plasmids from this publication at Addgene. Dynamic imaging of genomic loci...Oct 9. Pubmed. Find the plasmids from this publication at Addgene. ...

...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors show that...

...into this category describe how the user is expected to interact with the software. This category is important... CRISPR software submissions. With few prior publications to go on, a submitted manuscript could only ... solutions that facilitate most experimental applications in CRISPR engineering. When compared to that...easy to fault the lack of features in a single application. The question is then, should we use all tools...1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs. The second database...novo sgRNA design, I again separate it into two categories. On the one hand we have software tools that ...based on your project needs. There are 8 major categories found in the table, describing everything from...

...addgene.org Addgene's Antibody Purification Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody...cells can perform human-like post-translational modifications that bacterial and most yeast cells cannot. ...production system may depend on what downstream applications you are planning, cost, and the amount of protein...secretes fewer of its own proteins, making the purification process simpler. Due to its high yields, E. ...production (Schwarz et. al., 2020). Harvest and purification At its core, harvesting recombinant antibodies...your conjugate and conjugation chemistry.  Quantification Once you’ve collected your antibody in the ...accurate concentration to give your downstream applications the best chance at being successful.  Using ...

... inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins...technology, which uses locus-tagging and affinity purification, in 2009. However, iChIP requires insertion ... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic...describe this technology and some of its potential applications? Fujii: We generated a series of retroviral ... discoveries in biology. Therefore, my lab is dedicated to development of new biological technologies....interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences. Thus, we've made ...the peer-review system of journals and grant applications, for example. However, at the same time, it'...

...genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue...After self-excision, the resulting loxP scar is located in this synthetic intron, and only the fluorescent...use and highly efficient for C. elegans genome modification. Figure 2: Schematic from Dickinson et...permission from the Genetics Society of America. Applications of this system Dickinson et al. have developed...system could be used to make many other genome modifications, including targeted mutations. SEC-based systems... in cell culture. In addition to CRISPR/Cas9 applications, SEC selection could replace other, more restrictive... of print] PubMed. Find plasmids from this publication at Addgene. Other strategies for CRISPR/Cas9...

...the rest of the genome and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (...editing is most efficient when target sites are located in close proximity to the region to be edited. ... that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM sequences (Kleinstiver...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just as selective...to dramatically increase the number of CRISPR applications. The first non-Cas9 CRISPR protein adapted for...doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo T, Park SW, Kim D, Kim...

...playing a card game called “Magic”. There were complicated rules that governed how this game was played.... as I try being compelling, interactive, and application-driven to boost curiosity, I have never experienced...strategies work. Find Addgene Blog posts about other educational tools Playing BUILD YOUR PLASMID BUILD YOUR PLASMID... cards (Promoters, Inserts, Tags, Origins of Replication, Resistance gene and Selection Markers) and one...building, collaborative critical thinking, and communication among others. They additionally had to think...Who knows, perhaps the ideas spurred by this “gamification” will lead to new cures for disease someday!... each a variety of subjects at many different education levels. If you modify BUILD YOUR PLASMID or generate...

...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...to the selective nature of the cutting. While sonication is usually the preferred method, enzymatic digestion...concentration.  Protein Capture After crosslinking and sonication or enzymatic digestion, you’re ready to incubate...crosslinking. Finally, purify your DNA with a PCR purification kit or with a phenol-chloroform extraction.  ...

...function. You can also click one location in the plasmid and then a second location to select the sequence between...two separate locations in the linear sequence selects the sequence between the two locations and sends it...table includes feature names, locations, sizes, colors used to indicate them on the map, direction (if...promoters, origins, tags, and certain ORFs. Their sophisticated detection algorithm easily and precisely identifies...restriction enzyme annotations throughout the map at locations better aligned to their precise positions within...between the two locations and send it to the clipboard for copy and paste functionality. Sequence ...sequence. The table includes enzyme names, the locations where the enzymes will cut (in basepairs), and...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...characteristics, performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova...information on NIR FPs, their properties and applications. At the end, there is a Table that summarizes...photoreceptors incorporating biliverdin IXα (BV) autocatalytically. Fortunately for probe developers and users...vivo probes, due to low autofluorescence, low scattering, and deep penetration of red-shifted light. The...their enhanced versions named emiRFPs in your applications.  There is a whole set of spectrally distinct...of Medicine. Her expertise is development and applications of fluorescent proteins, genetically encoded...

...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... carboxylase and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...uses of biotinylation in the lab are protein purification and immunoprecipitation experiments. Biotinylated...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...bound to your protein of interest leading to amplification of the reporter signal and easier detection....

...current CRISPR research focuses on its potential applications for human medicine (Waltz, 2016), the potential...engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a ...model organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn...engineering using CRISPR, highlight the specific modifications to CRISPR machinery that allow for the use of...CRISPR principles). However, plant-specific modifications to commonly used CRISPR plasmids are necessary...selected the correct CRISPR components for your application, it is time to deliver these components to your...detergent, Polyethylene Glycol (PEG), although the application of this approach is limited to protoplast cells...

...labeling can be subdivided into two categories, depending on the location of the targeted markers. Some commercially...proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation patterns...Depending on the research question and downstream application, one or several of those techniques can be used...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...labeling Oftentimes, a marker of interest is not located on the cell membrane but inside the cell. Due to...but preserving the cellular state (think of mummification). Permeabilization, as the name suggests, perforates...the nucleus intact. If markers of interest are located inside the nucleus, a different fix/perm kit is...

...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...that fall into this category include performing experiments with too few replicates leading to suspect ...these positive results will be published. The publication record will then show that the tested hypothesis...reproducible publishing practices. One example is the publication of peer reviews so we can see how and why papers... also means that you’re more likely to get a publication (for better or worse, the currency for success...review of experimental design as part of the publication process (Nosek et al 2018). In this ideal situation...

...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...observed to exist in various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization...other methods below should help in multimer identification.   Fig. 2: Gel image of Plasmid 61564...within a plasmid and, therefore, allow for the identification of plasmid multimers. Capillary Gel Electrophoresis...multimerization. One study by Bacolla et al. (2011) indicated that strain HB101 produced the highest yield of...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR efficiency...PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et ...PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh...