We narrowed to 513 results for: gats

... 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...

... 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...

...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...

...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  ...

...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...

... 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...

...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...

...alternatives to the standard restriction/ligation, Gateway, or Golden Gate cloning methods. Whether it is for...based technologies We've described Sequence and Ligation Independent Cloning (SLIC) in a previous Plasmids...polymerase extension cloning) and SLiCE (Seamless Ligation Cloning Extract) to name two more. Likewise, there... work by significantly different methods. Golden Gate cloning does result in the seamless joining of fragments...location and NOT in the middle of your insert. Gateway cloning utilizes λ integrase to catalyze directional...

...Positive and negative controls Most biological experiments require positive and negative controls to ensure...experiments will benefit from positive (+) and negative (-) controls in addition to experimental samples...In this case, mouse samples from positive (+), negative (-) and experimental (?) groups were run on a ... check for tau molecular weight shifts. A negative control is a sample or group that is not subjected...experimental condition. In the example above, a negative control could be a third group of mice that is...receive either protein kinase inhibitor. Since the negative control group does not receive the drug, any response...

...fluorescently-conjugated streptavidin to get more fluorophores per antibody, or conjugate with an enzymatic...primary antibody to bind the target, wash, add dye-conjugated secondary antibody that recognizes the primary...do simultaneous or sequential labeling is what conjugated secondary antibodies you have available. If the...only secondary antibodies you have on hand are conjugated with the same dye (say, you have Goat Anti-Rabbit... direct IF, using primary antibodies that are conjugated to fluorophores. However, the primary-secondary...or scFvs. Many, many options for fluorescently conjugated secondary antibodies are commercially available...

...process consisting of digestion, purification, ligation, and transformation. While restriction cloning... developed. Some of these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning...developed by Li, et al., in 2011. This method is ligation independent, it does not require purification ...Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation + + The FastCloning process... reactions. These sticky ends are annealed and ligated in vivo. Detailed FastCloning procedure To start...highly simplified, purification-free, sequence-and ligation-independent PCR cloning method." BMC biotechnology...

...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)...

...dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright, & Ebright, 2001). These Cy/NTA conjugates work the same ...tag from 6 to 10 residues. While these Cy/NTA conjugates are not commercially available, they are easily...in intact cells. The Gradia Lab has deposited a gateway vector with Addgene, where you can insert your ...adjacent to each other on the same protein and a negative control experiment conducted on donor-only labeled... (Ni2+: nitrilotriacetic acid) n-fluorochrome conjugates." Journal of the American Chemical Society 123.48...Your Experiments Beware of Fluorescent Protein Aggregation Brush up on Your Fluorescent Protein Basics ...

...gRNAs. As each ligation reaction uses a pool of gRNAs as starting material, the ligation products are a... et al., 2014) individual genes for studies interrogating their functions. The continual advancements ...with straightforward restriction digestion and ligation steps to build barcoded lentiviral plasmids containing...lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid libraries containing combinations... developing and applying new technologies to interrogate and understand complex biological systems. He...

...transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell to another). Bacteria...need special fertility genes to be capable of conjugation, and these are encoded by certain kinds of plasmids...to a recipient through direct contact. That’s conjugation! F factors can also be integrated into the bacterial...also have more than one function: for example, Gateway cloning relies on genes found on an E. coli fertility...contain this F plasmid is critical for successful Gateway cloning. In addition, scientists have engineered...101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org Addgene's...

...though most MoClo reactions use Golden Gate cloning, not all Golden Gate cloning is MoClo. That’s because MoClo...Golden Gate Assembly with NEB’s DAD Interview with Nicola Patron on MoClo Plasmids 101: Golden Gate Cloning...hierarchy. MoClo originally developed from Golden Gate Cloning, in which a type IIS restriction enzyme ...MoClo that relies on Gibson and other non-Golden Gate cloning methods. MoClo’s organization schema MoClo...

...incomplete lysing/insoluble proteins, and protein aggregation. Protein degradation, which would result in low...from their binding. If you’re seeing protein aggregation, you may want to consider changing your incubation...incubation temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a longer...incubation (30–60 minutes) at 37 °C, if you suspect aggregation in your prep. If your proteins are the wrong ...binding site, allowing you to visualize them with a conjugated antibody. If you do not need such precise measurements...of alcohol. The increased SDS will increase the negative charge on the protein, allowing it to move more...Blot Doctor page, or robust discussions, like ResearchGate’s western blot forum.  Blocking Don’t skimp on...

...it allows for better resolution of positive and negative populations, Careful panel design In both conventional... your fully stained samples. If you are using conjugated dyes, it is critical that your controls and your...variation. Tandem dyes (two fluorescent molecules conjugated together) can be especially sensitive to batch...batch effect. For example, when a PE molecule is conjugated with a Cy7 molecule, there is no telling exactly... (positive population) or won’t bind anything (negative population). It is strongly recommended that when...AntibodiesAntibodies 101: Buffers, Storage, and Conjugates Resources on Addgene.org Addgene's Antibody GuideAddgene's...

...to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]. This is a problem ...localization, dynamics, and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce...has gone into making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally ...and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several cell proteins normally... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...moderately dimerizing FPs such as EGFP, but not for obligate oligomers such as DsRed or high affinity FPs such...

...of the first BiFC studies used the method to interrogate the subcellular localization of interactions ... may also work. You will also need to generate negative control constructs — more on this later. For more...fluorescence intensity of your BiFC constructs to your negative control to determine whether your PIPs actually...actually do interact. Choosing the right negative control is critical to the success of BiFC experiments. The... The FP fragments themselves cannot be used as negative controls because their expression level, stability...BiFC competition analysis can be used in lieu of negative controls (Kerppola, 2006). Here, one of the endogenous...