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...adding different ligands to the same sample to label sub-populations of your protein of interest. For example...range of useful molecules linked to that substrate. If the substrate was linked to, say, a chemical dye or...instead become trapped in a covalent bond with its substrate. Then, all it takes is a bit of chemistry magic... gets covalently linked to derivatives of its substrate O6-benzylguanine (BG) (Keppler et al., 2003). ...be labeled with fluorescent derivatives of its substrate trimpethoprim (TMP-tag) (Miller et al., 2005)...., or other uses of your protein, such as with substrate-linked PEG, biotin, or magnetic beads. In one ...

...of techniques have been developed that allow for sub-diffraction limit imaging. Most often, these techniques...number of optical and chemical ‘tricks’ to turn subsets of fluorescent molecules on and off. This can provide...-time monitoring of neuronal activity across substantial 3D volumes. In addition to 2-photon microscopy...

... the plant-specific parts you‘ll need, including sub-cellular localization signals, plant markers, and...

...genes, and is unique in that it is composed of two sub-pools. The first is a base “90k library” that contains...using CRISPR-X and subsequent FACS sorting. They also mutagenized proteasome subunit PSMB5 to find mechanisms...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...mRNA binding, and these sequence changes are subsequently identified through transcriptome sequencing.... Guler Lab engineered an actuator in which two subunits of the paramagnetic ferritin protein were tethered...channel). The Guler lab optimized the actuator’s subcellular localization using trafficking signals and named...

...secretory proteins or proteins targeted to membrane sub-domains. It has also been used to observe intra- ...Renilla luciferase, catalyzes the oxidation of its substrate, luciferin, which releases energy as a photon.... shown to be useful tools for light-mediated subcellular localization in mammalian cell culture and reversible...were used in a variety of experiments including subcellular visualization of 6 different organelles, a cell...photoactivatable fluorescent proteins (FPs). A subpopulation of mitochondria is selectively labeled by irradiating...assaying the dynamics of different mitochondrial subpopulations within the same cell; by photoswitching PSmOrange...PSmOrange and photoactivating PAGFP in separate subpopulations of mitochondria, one can monitor how the two...

...deposit process by clicking "Submit Online Deposit" (for online submission) or "Download Deposit Spreadsheet...emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...   Figure 3: If submitting plasmid(s) online (left), choose to submit as published or unpublished...page and click the blue "Submit Deposit" button. Figure 13: Click the "Submit Deposit" button to complete...begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a spreadsheet. The...Fields marked with an asterisk are required. If submitting plasmid(s) using a spreadsheet (right), click...download the .xls file. For the online form you can submit "Published plasmids" or "Unpublished or pre-publication...

...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...   Substrate DNA Design and Generation Whether you use a linear dsDNA or ssDNA substrate depends ...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...Pro Tip: ssDNA substrates have a much higher rate of recombination than dsDNA substrates, so you should...DNA, you first electroporate a linear donor DNA substrate (either dsDNA or ssDNA – see below) into E. coli... catalyze the homologous recombination of the substrate with the target DNA sequence. This means cloning...genetic changes occur in a test tube. The donor DNA substrate only requires ~50 nucleotides of homology to the...

...blog post shows. To avoid these issues, Subach et al. and Tsuboi et al. developed monomeric FP timers with...monomeric FP timers (mK-GO from Tsuboi et al. and the FT series from Subach et al.) were derived from previously...membrane protein, and following its subcellular localization overtime (Subach et al.) Monitor protein expression...tetramer with a propensity to aggregate in cells (Tsuboi et al.), limiting its use. Nonetheless, its creators...fluorescent proteins mKO and mCherry, respectively. While Tsuboi et al developed mK-GO somewhat serendipitously ... while attempting to enhance mKO in other ways, Subach et al made a concerted effort to develop their ...see Table 1 for emission and absorption spectra). Subach et al’s directed mutagenesis also produced the ...

...membrane and deliver the substrate; however, secreted luciferase or alternative substrates can permit measurements...incompatible substrates can also allow multiplexing with experiments that supply both substrates and measure... Bioluminescent reporters require a chemical substrate (and sometimes cofactors) to be provided and are... they’ll keep emitting light as long as their substrate is available. Table 1 compares some general advantages...of photon generation Slower Faster Cofactors/substrates Required Not required Signal strength Lower.../phototoxicity Not susceptible Susceptible Subcellular imaging Improving Well-established High-throughput...These variant proteins offer different kinetics, substrate requirements, and photon emission wavelengths....

... out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar compositions and...immunity. Within type III, there are six subtypes, A–F. All subtypes use Cas10 as the primary effector complex...into a fairly simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps...types are based on main function and composition; subtypes and variants are typically based on species of...There are currently two classes, six types, 33 subtypes, and 17 variants. Figure 1: Overview of...hierarchy. The hierarchy flows from Class to type to subtype to variant (shown in italics). Created with BioRender.com...Figure 2: Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to ...

...different subclasses, but there are also secondary antibodies that are specific to each subclass. Approximately...mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass specific secondary...backbone to generate R-mAbs of different subclasses, including subclasses that were not produced by the original...original hybridoma. By using subclass specific secondary antibodies and subclassed switched R-mAbs, multiple... by some of the hybridomas and, as a result, subsequently cloned into the expression constructs. The Trimmer...engineered. Like humans, mice express multiple subclasses of IgG antibody. Broad range secondary antibodies...  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain...

...unique antibodies that bind discrete subunits or particular subunit pairs. Some of the antibodies in the... one of 18 alpha and one of eight beta subunits. Both subunits have short intracellular ends and large...region displayed by a subset of the RGD-binding integrins containing the αV subunit. Using these affinity...discovered by yeast display reveal αV subunit pairing preferences with β subunits (p. 2024.01.26.577394). bioRxiv...well-known motif serves as a recognition site for one subfamily of integrins, the RGD-binding integrins. Six of...remarkable specificity for their targeted integrin subtype, even when in competition with other similar integrins...antibodies to show the binding preferences of the αV subunit and its β partners. Of interest, we demonstrated...

...three subunits: alpha (Gα), beta (Gβ), and gamma (Gγ). G proteins are divided into four subtypes, based...associate with certain subtypes, although most are able to signal through more than one subtype. How GPCR signaling...disassociation of the Gα subunit from the Gβγ dimer. The Gα and Gβγ subunits then induce or inhibit intracellular...based on their Gα subunit: Gαs, Gαi/o, Gαq/11, and Gα12/13. Gβ and Gγ are closely associated and function...change in the GPCR causing the activation of the Gα subunit. The activated Gα exchanges bound GDP with GTP,...inactive heterotrimeric G protein complex and subsequently another round of signaling.    Addgene Tools...plasmids encoding 14 alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G protein complex that can...

...influence your choice of journals to submit your work to. Before submitting your paper to your preferred  ...approve your paper. Rush your submission or revision. A sloppy submission, missing files or a manuscript...scientific community, or are you writing for a specific subgroup of researchers in your area of expertise? Sometimes...who wishes to read it, without them needing a subscription to the journal. An increasing number of funding...a reliable source of information, it is best to submit it to a journal that uses a peer review system....if you don’t. Add a clear cover letter to your submission, briefly summarizing the conclusions of your ... the Author Guidelines of the journal you are submitting to and make sure your manuscript fits the house...

...community submissions for Addgene and CLOVER AAV experiments - and we've updated our submission process ...say, should!) submit your data to the Data Hub! The process is easy with our new submission form - and no...step-by-step submission guide or email [email protected] to learn more. A great submission includes: ... cited by others?)  Ready to submit?  Fantastic! The next 25 submissions will receive a 20% off coupon...recording to see two reports presented by the submitting scientists, learn more about the AAV Data Hub...given by Connie Cepko and Ian Wickersham.     Submitting to the Data Hub If you've ever validated an Addgene...

...0.02–0.05 ml Skill required: beginner Subcutaneous Subcutaneous (SQ or SC) injections are given between...method, subcutaneous injections take some practice to get the coordination down. Subcutaneous injections...the mice, and restraint method. Many different substances can be injected, including cells, drugs, DNA,.... IV injections are used for a broad range of substances, as it circulate what is injected throughout ...technique. Intrathecal injections are used when the substance being administered must be delivered to the central...Intratracheal instillation is used to deliver substances directly to the lungs. It is an advanced technique...invasive, and only requires a pipette. Since the substances are administered through the nose, they also ...

...neuronal subtypes was not available until now. In order to target specific neuronal subtypes such as inhibitory...recently deposited 2 Bacillus subtilis libraries with Addgene – the B. subtilis Single Gene Deletion Library—Kanamycin...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...hot plasmids segment on the Addgene Podcast. Subscribe to the Addgene blog to get all the latest hot ...containing high fidelity Cas9 variant HF-Cas9. They subsequently tested the original BE3 and HF-BE3 using a gRNA...the podcast Segement on the pPSU Plasmids B. subtilis knockout and knockdown libraries The Gross Lab... Essential gene Knockdown library. Although B. subtilis has a fully sequenced genome, many applications...

...are not subject to photobleaching/phototoxicity, they successfully imaged multiple subcellular compartments...luminescent substrate is required for imaging, and it is important to verify that this substrate does not...fused to an FP; when supplied with a luciferase substrate, the luciferase transfers energy to the FP, resulting...excitation light source. Instead, a luciferase substrate like coelenterazine is supplied in the culture... which is limited by depletion of luminescent substrate, they synthesized diacetyl coelenterazine-h. Unlike...coelenterazine-h, producing a constant supply of substrate. Using this compound to excite the Nano-lanterns...brightest characterized luciferase, NanoLuc, and its substrate furimazine. In addition to the previously described...

...ligate many undesirable structures, including substrates containing gaps or DNA base pair mismatches (...such as Taq DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments... sequence is present, the probes will ligate. Subsequent cycling through rounds of melting and annealing...generally prefer fully Watson-Crick base-paired dsDNA substrates to those containing one or more mismatches. However...pyrimidine mismatches are typically worse ligation substrates than pyrimidine:purine mismatches. Helix stability...crystal structures of several DNA ligases bound to substrate (23, 24). Thermostable DNA ligases, including ...naturally able to discriminate against ligating substrates containing base pair mismatches (i.e., are "higher...

... have subscriptions to the specialized journals in your field? Post a preprint before you submit to a ...article, presenting a conference poster or talk, or submitting patent or grant applications; to informal settings...especially the increasingly growing time from submission to publication. It takes many months, often more...the possibly repeated rounds of rejection and resubmission to other journals. Preprints allow authors to...admittedly, somewhat meaningless) practice of listing “Submitted” articles in their CV or grant applications. A...post a preprint at the same time as, or before, submitting the manuscript to a journal for formal peer review...opportunity to strengthen the manuscript prior to submission. Preprints at Addgene Preprints are all about...