...generated against a specific antigen and then conjugated with a fluorescent molecule allowing one to “...secondary antibody that targets the primary is conjugated to a fluorophore (Figure 1).   Figure ...you can begin to see why generating fluorophore-conjugated versions for each primary antibody could get ...secondary antibodies that target common IgGs and then conjugate them with an array of fluorophores. It is common...then use the corresponding secondary antibodies conjugated to distinct fluorophores to visualize multiple...apply, which binds to the first antibody and is conjugated to a fluorophore. You may need to optimize the...or have been manipulated to overexpress it. For negative controls, use samples in which the target is known...

...-RP4 conjugation system. This system can deliver plasmids to both Gram-positive and Gram-negative cells...Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC). With MAGIC, scientists introduce an ...phyla that have taken up the plasmid through conjugation in vivo studies. However, they only identified... of modular parts that are assembled with Golden Gate Assembly. They built this toolkit upon the Yeast...retains the oriT for delivery into recipients by conjugation. The labs screened for the host-range of the ...fluorescent proteins into one bright particle that segregates into one cell during cell division. To achieve...PMC5963681.  Meyer, Richard. "Replication and conjugative mobilization of broad host-range IncQ plasmids...

... is directly conjugated to a detection substrate, such as a fluorophore, or a conjugated secondary antibody...a protein in a tissue section then a directly-conjugated antibody will provide better resolution as these...immune complexes are smaller than a primary:secondaries:conjugates complex. If you are trying to visualize... Some applications use either direct (primary-conjugated) or indirect (secondary) methods. In those cases...accomplished by using a panel of primary antibodies conjugated to different fluorophores or a panel of primaries...species, then you can choose different fluorophore-conjugated secondary antibodies specific to each isotype...troubleshoot and be sure to include proper positive and negative controls as you get your assays up and running...

...Addgene: And the BIL gates collection? Endy: The Boolean Integrase Logic (BIL) gates collection of parts...switches that enable cellular computing. These logic gates built from transcriptors are now available at Addgene...Addgene spoke with Endy more about the BIOFAB and BIL gates plasmid kits, the Biobricks Public Agreement, and...bioeconomy. Addgene: What do these BIOFAB and BIL gates kits – the first to be made available via Addgene...systems, plumbing, computers, and now through BIL gates we've implemented them in DNA. We thought these ...work, watch Endy's 10-minute YouTube primer on BIL gates.) Addgene: Why is it so important to you that these... Jerome Bonnet et al. "Amplifying genetic logic gates." Science. 340, 599-603 (3 May 2013). Vivek Mutalik...

...or the primer design. Use a negative control strain. A good negative control strain is an untransformed...with a positive clone amplifying a product and a negative clone resulting in no product. Additionally, this...clone will produce a larger size product then a negative clone without the insert. This type of primer ...on the number of background colonies on your no ligation control plate. The more background, the more colonies... positive clones (+) will give a band, while a negative clone (-) will not. Backbone-specific primers ...sized products for positive clones (+) compared to negative clones. Finally, orientation-specific primers ...important for insert-specific primers. If your negative control amplifies a product of the expected size...

...transcription. The HIS3 reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu...screens are prone to both false positive and false negatives, these experiments must be set up, conducted and...leads with other methods. More common are false negatives; up to 75% of known interactions can be missed...partners with other techniques! Minimizing false negatives: Interactions leading to detectable reporter signals...parameters can lead to a high number of false negatives in Y2H screens. Testing your reporter system ...protein doesn’t function in yeast! Many false negatives stem from or are exacerbated by expression of ...expression levels and systems can alleviate false negatives that arise from low or faulty expression of your...

... the same name. The tag has a net negative charge with 4 negatively charged and 1 positively charged amino...hemagglutinin. The tag has two negatively charged amino acids for an overall negative charge. This tag has been...common tags, FLAG is the most charged, with 5 negatively charged and 2 positively charged amino acids....localization and solubility, so be on the lookout for aggregation of your protein of interest!   Ready to start...Arribere, J. A., et al., Translation readthrough mitigation. Nature. (2016) 534, 719-723. DOI: 10.1038/nature18308...

... 84,963 Human CRISPR Knockout Pooled Library (Gattinara) 136986 Knockout Human Doench 3rd 2 40,964 Ingolia...

...outside their field, the answers were primarily negative. Stupid, overwhelmed, discouraged, tired…words...from AAV Purification by Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader...for AAV Purification by Iodixanol Gradient Ultracentrifugation. The reader may be, for example, a graduate...between a gradient tube before and after ultracentrifugation so that a scientist can extract virus from...interfaces between layers are blurred after centrifugation and the syringe needle is inserted bevel-up...profile photo of an iodixanol gradient after ultracentrifugation. The interfaces between layers are muddled...illustrates a gradient tube before and after ultracentrifugation. The viral supernatant migrates through increasingly...

...Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent cloning, restriction...Please indicate which growth strain Addgene should propagate your plasmids in: DH5α, NEB Stable, ccdB Survival...Survival, Pir1, or other. Whenever possible, we propagate plasmids in the standard cloning strain DH5α. For...For plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids...choose the Pir1 strain. If your plasmid cannot be propagated in any of these standard strains, choose other...Distribute pending QC. The name of the Principal Investigator and Organization where your plasmids were first...

... antibody is also conjugated to another molecule, such as biotin. Next, a conjugated substrate is added... biotin-conjugated, streptavidin (which has a high affinity for binding biotin), is conjugated to the ...protein of interest is then detected by an enzyme-conjugated antibody. Next, its substrate is added and the...instead of detecting your protein using an enzyme-conjugated antibody, a primary-secondary pair is used. This...single primary antibody. The secondary antibody is conjugated to an enzyme for visualization. The indirect ...

... by ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus...vectors) you can concentrate your virus by ultracentrifugation of the viral stock, followed by resuspension...packaging cell debris before proceeding to ultracentrifugation to avoid contaminating your transduction ...through a 0.45 µm or 0.22 µm filter) or by a centrifugation step (5 minutes at 300-500 g). After removing... to concentrate a virus solution without ultracentrifugation, you can reduce the volume of cell culture...repulsive electrostatic forces between these two negatively-charged membranes (Davis et al., 2002). Polybrene...

...components: It's important to assess how dye-conjugation affects protein function and its propensity to...process: The process of dye labeling, such as conjugation with proteins, should be carefully managed. Proper...degree of labeling (DOL), and thorough removal of unconjugated dye molecules are critical for accurate and ...assay results (Hayashi-Takanaka et al., 2014)​​. Conjugates with lower DOL tend to have weaker fluorescence...fluorescent dye. For instance, the cleavage of FITC-conjugated protein ligands is unaffected by additional protein...non-specific binding, and quenching, which can be mitigated through careful selection and optimization. Fluorescent...Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging. PLoS ONE, 9(9)...

...detected using an unconjugated primary antibody, then visualized using a fluorophore-conjugated secondary antibody...Antibodies - typically, primary antibodies that are conjugated to a fluorophore, to avoid the use of secondary...the signal comes from the antibody-fluorophore conjugate, and is not expressed by the cell itself, there...lot of flexibility in which proteins you can investigate in each panel. An advantage of flow cytometry.... These could be visualized using fluorophore-conjugated antibodies (i.e., immunofluorescence), or by .... These applications are typically used for investigating signaling pathways or developing therapeutics...therapeutics. Experimentally, antibodies conjugated to fluorescent tags or other markers can be injected into an...

...all the latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox for studying the nervous system...system The Cole lab recently deposited the Tol2 Gateway-Compatible Toolobox, which will allow scientists...neurobiology and neuorodgenerative diseases. The Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit...empty vectors that have standard cloning sites or gateway compatible cloning sites for easy cloning of your...toolbox adds new neuronal tools to the expanding gateway compatible vectors currently shared amongst the...2007. PubMed PMID: 17937395 Listen to Our Tol2 Gateway-Compatible Toolbox Podcast Segment The impact of...to premature aging and cancer. The Denchi lab investigated an aspect of telomere length regulation called...

... a specific conjugate, and changing your reporter molecule requires either re-conjugating or re-purchasing... while providing flexibility in assay design. Conjugated secondary antibodies are relatively cheap and...westerns typically rely on primary antibodies conjugated to fluorescent proteins, instead of the more ...the enzymes used for chemiluminescence can be conjugated to primary antibodies). Direct westerns are simpler...or loading sample, which may not be practical. Conjugated primary antibodies can be more difficult to source...antibody with the new reporter. Finally, each new conjugate will have to be tested to ensure it doesn’t impact...if you prefer the DIY method. As some proteins aggregate at 95 °C, you may want to check for any available...

...constructs with Golden Gate MoClo. The barrier for those switching to Golden Gate is a conceptual barrier...synthetic biology Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include...people already use. It makes the switch to Golden Gate Cloning easier because individual labs will not ...soon to make it easier for people to use the Golden Gate cloning system especially with plants in mind. When... work in cancer biology.  References: A Golden Gate Modular Cloning Toolbox for Plants. Carola Engler...Reading: Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

... fluorophore conjugates are used, but these are usually more expensive than unconjugated primary antibodies...cysteine. Maleimide-conjugated HRP can be found here and several maleimide-conjugated dyes are also commercially... primary antibodies with secondary nanobodies conjugated with the desired fluorophores. In figure 4B below...individually incubated with a secondary nanobody conjugated with Alexa Fluor 488, Alexa Fluor 568, or Alexa...cysteine sites the nanobody has available for conjugation to a specific label (see step 4 for more information...

...restriction enzyme digestion followed by ligation. Upon ligation, the original pattern of the MCS is regenerated...of the ligation product, and the restriction site between the modules is eliminated upon ligation. As a...regenerated at the flanks of each ligation product, which is the basis for the repeatability of the method...added to the 5’ terminus or the 3’ terminus of the ligation product. Comparison to other high throughput cloning... Method Technology Pros Cons References Golden Gate Type II restriction enzymes Can assemble up to 10...

...microscopy after negative staining. Empty vector particles can be identified after negative staining and ...preparations are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate...final vector preparation, the quantity of gram-negative bacterial endotoxin is ensured to be less than...containing lentivirus is first cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone...in 10% polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles...possible, all plasmids used for viral production are propagated in the endA-mutated NEB Stable strain of E. coli...