...transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements...antibody applications. Intro to the Lab Bench Name Description (Link opens in a new window) Link to Video Personal...materials on a balance Basic Molecular Biology Name Description (Link opens in a new window) Link to Video Making...without a commercial kit Plasmid Cloning Name Description (Link opens in a new window) Link to Video Restriction... targeting human and mouse genes. Virus Name Description Link to Video General Transfection Introduce ... your lentivirus preparation Antibodies Name Description Link to Video Transfection Introduce plasmid ...

...This protocol describes the basic steps for lysing cells, determining total protein concentration in ...specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining ...Nonfat milk powder 96-well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary... at RT on a shaking platform. Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent...reagent B. Gently incubate the membrane in the chemiluminescence reagent for 5 min at RT . Cover the membrane...with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands...

...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...Protocols Viral Service Introduction This protocol describes how to generate a monoclonal cell line from a ...type will be able to grow under the conditions described in this protocol. Conditioned medium (details ... specific conditions used. Each 96-well plate described in this protocol could, in theory, give rise to...protocol was performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise...from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved vectors and genome-wide libraries ...

...amplification of CRISPR pooled plasmid libraries in Escherichia coli cells....amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to ...activating pulse and follow all specifications described in the equipment manual. Immediately add 1 mL ...µl aliquots of cell/DNA mixture used (8 in the described protocol) using a new vented falcon tube for every...different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than ...

...Protocols Viral Service Introduction This protocol describes a general method for transfecting mammalian cells...with fresh media Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment Equipment...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...efficiencies as can be seen here by the amount of green fluorescent protein expression (green in the right panels...

...Protocols (Link opens in a new window) Video Link Description Related Page Making LB Agar Plates Create plates...Videos (Link opens in a new window) Video Link Description Related Page MTA Guide - Requests Your guide ...Videos (Link opens in a new window) Video Link Description Jessica Welch, PhD In this first installment ...

...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected...Protocol Antibody Guide Introduction This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent...antigen to validate an antibody. This protocol will describe how to create a standard curve using known concentrations...

... Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene...Guide Virus Blog Posts Introduction This protocol describes how to use droplet digital PCR (ddPCR) to titer...and titrations Reference Wang Y, Bergelson S, Feschenko M. Determination of Lentiviral Infectious Titer...

... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension onto the agar and gently spread...

....9kb stuffer, visit www.addgene.org/10878/ . Description Vector Element U6 Human U6 promoter drives RNA...the pLKO.1 TRC-cloning vector. Addgene Plasmid Description pLKO.1 – TRC control Negative control vector ...license or rights of access or use to the materials described in the literature. The distribution of materials...

...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

...This protocol describes how to affinity purify recombinant antibodies from cell culture supernatant using...Western Blot Protocol Introduction This protocol describes how to affinity purify recombinant antibodies ...

...exchanging the fluorescent donor for a bioluminescent donor, making BRET probes. Here the bioluminescent donor ...NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...better biosensors. NanoLuc®-fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging...more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited ...transfer of energy from one fluorescent protein to a compatible fluorescent protein. FRET can be measured...activation of other fluorescent probes but can still transfer energy to the fluorescent protein acceptor....these tools, NanoLuc-fluorescent protein fusion shifts NLuc’s bright blue luminescence to wavelenghts more...

...such as fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy... have a variety of fluorescent light sources. When interrogated with a fluorescent light source, a cell...conjugated fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by ...laser and interrogated with various visible and fluorescent light sources that allow users to assess a cell... cells in a population in a procedure termed fluorescence activated cell sorting or FACS (2). By analyzing...for your study see Addgene’s blog post Which Fluorescence Microscopy Techniques is Best for Me?  An overview...particular surface receptor are stained with a fluorescent antibody against the receptor and analyzed on...

...chromoproteins, fluorescent proteins, and luminescent proteins. While fluorescent proteins and luminescence have ...is full of striking visuals from fluorescent jellyfish, bioluminescent phytoplankton, and colorful corals...from fluorescent proteins? Before going into the difference between chromoproteins and fluorescent proteins...the widely used green fluorescent protein (GFP) gives a bright green fluorescence when exposed to blue ...light, whereas chromoproteins do not fluoresce at all or fluoresce at very low levels. Chromoproteins in...for instrument-free detection. Unlike fluorescence or luminescence, which require UV lamps, fluorometers... chromoproteins are actually a subset of the fluorescent protein family. What sets them apart is the way...

... blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org Fluorescent protein...area with lots of bright new plasmids is our Fluorescent Biosensors Collection — scientists are always...measure! Let’s take a look at some of the hottest fluorescent biosensors new to Addgene this year. Feel the...Hiroshi Hibino and colleagues devised a “Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular...region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent protein is inserted into one of the loop regions...  Find MTRIA sensors here!   Ino D, et al. A fluorescent sensor for real-time measurement of extracellular...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific...

...than any fluorescent protein earlier described. “Until our proteins, there were lots of fluorescent proteins...increase their fluorescence upon illumination with far-red light. As the researchers described in Nature Communications... If you start poking around on Addgene’s Fluorescent Protein Guide to In Vivo Imaging, you’ll pretty ...good reason. We all know scientists have used fluorescent proteins to observe what’s happening inside cells... of decades. Green is the classic color, but fluorescent proteins are available in a variety of hues. ...unimpeded. The challenge then was to develop fluorescent proteins that would fall within that near-infrared... from jellyfishes and corals, but all of them fluoresce outside the transparency window of mammalian tissues...

...More about Fluorescent Protein-Based Tools Learn Some of the Pitfalls When Using Fluorescent Proteins Control...Resources on Asddgene.org Find Fluorescent Protein Plasmids Find Fluorescent Protein Kits Find Empty Backbones...historically been difficult, as techniques like fluorescent in situ hybridization (FISH) and chromosome capture...be used in vivo. Some researchers have used fluorescently tagged DNA-binding proteins to label certain...chromosome. This image represents the composite of fluorescent measurements from red, blue, and green channels...targeting capacity. By using a gRNA to target fluorescently labeled dCas9 to a given genomic locus, you ... thereby decreasing the amount of background fluorescence from unbound GFP-dCas9. With these improvements...

...simultaneously acquired from the the fluorescent and luminescent siganls and a ratio is calculated (the...26456817. Nano-lanterns for multicolor luminescence imaging  Fluorescent imaging techniques have become indispensable...visualization of single molecules via targeted fluorescent molecules. The Vale lab and the Weissman lab ...protein of interest are recognized and thus fluorescently labeled by scFv antibodies fused to sfGFP. This... This system amplifies the intensity of fluorescence signal and enables tracking of single molecules within... plasmids to increase the brilliance of your fluorescent signal and endogenous activation of your target...Dec;11(12):1261-6. doi: 10.1038/nmeth.3147. Bioluminescent pH sensor Understanding intracellular pH regulation...

... of fluorescent fusion protein with your gene of interest: Plasmid Color Expression Description pPROEX... filters chosen for the donor fluorescence and the acceptor fluorescence. Additionally, this method could... the fluorescence decay time of the donor. When FRET occurs between the pairs, donor fluorescence is quenched...factors, such as pH or autofluorescence background, can change the fluorescence decay time and have to...Master. Note: BRET= Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer...course talking about the now well-known FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique...: the acceptor photobleaching method and the fluorescence lifetime imaging microscopy (FLIM) method. Acceptor...