...primary antibody against a target protein and a fluorescent secondary antibody....primary antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the... tips and pipettes Rocking platform Tweezers Fluorescent microscope 0.5–10 µL single channel pipette 2...500 µL PBS on a rocking platform. Dilute the fluorescently-labeled secondary antibody to the desired concentration...generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary antibody to the wells and ...cell labeling on a microscope with appropriate fluorescent filters. Tips and Troubleshooting The optimal...

...maintenance phase and do not allow cells to reach 100% confluence (80–90% is ideal). Pass and plate the cells the...from 2 x T-175 flasks. Cells should be at ~80% confluence. For each T-175 flask: Aspirate culture media...resuspend cells from the CS2. Cells should be at ~80% confluence. Aspirate culture media and rinse once with 60...Sample Data Figure 1: HEK293T cells at various confluencies. This AAV production protocol should be started...started with cells that are ~80% confluent (center panel). These cells were imaged with a 10X objective ...

...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...

...the individual wells become confluent, expand into larger vessels. A confluent well of a 6-well dish can...can be expanded into a 10 cm dish. A confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc....

...the next day they will be approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in...Do not use the medium if the cells are overly confluent or the medium is acidified (i.e., the medium appears...cells have expanded but before they become over-confluent, trypsinize the cells and expand them to larger...

...cells from incubator. The cells should be 50-80% confluent and in DMEM that does not contain antibiotics....The target cells should be approximately 80-90% confluent. c. Dilute puromycin in the preferred culture ...: b. Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg...

...transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements...

...ROX dye), to normalize non-PCR–related fluorescence fluctuations and to minimize well-to-well variability...

...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...

...different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than ...

... Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene...

... curve with known amounts of protein. Fluorophores Fluorophores are light-sensitive molecules which emit...cell or tissue sample. Figure 6: Immunofluorescence, in which fluorophore-conjugated primary antibodies ...label cells in a sample. Immunofluorescence (IF) This method uses fluorophore-conjugated antibodies to...IF uses fluorophore color to differentiate between the targets of interest, if your fluorophore is bound...wells) by fluorescence activated cell sorting (FACS). Figure 7: Flow cytometry uses fluorescently-labeled...the fluorophores used is important. It is best to inversely pair the brightness of the fluorophore with...If a bright fluorophore is on an abundant target, it obscures fainter signals. Fluorophores can emit signals...

...genome encoding Cre-dependent expression of the fluorescent protein EYFP from the CAG promoter Viviana Gradinaru...An AAV genome that ubiquitously expresses the fluorescent protein eYFP from the CAG promoter Viviana Gradinaru...Cation channelrhodopsin ChrimsonR fused to mRuby2 fluorophore and targeted to the neuronal soma and proximal... AAV production plasmid encoding for Archon1 fluorescent voltage reporter, Cre-dependent expression Edward...

...Photoconvertible , and Photoswitchable fluorescent proteins Various PIs Fluorescent protein fusion: contains empty...with N and C-terminal fluorescent protein tags that can be used to create fluorescent protein fusions with...cyan fluorescent protein variant (CyPet) optimized for FRET measurements with a yellow fluorescent protein...yellow fluorescent protein variant (YPet) optimized for FRET measurements with a cyan fluorescent protein...pRSETa mEos2 20341 mEos2 (fluorescent protein) Loren Looger Photoactivable Fluorescent Protein Expression: ...blot. The plasmids in this collection contain fluorescent protein tags whose localization you can monitor...Technique/Tag PI Purpose Empty Backbones with Fluorescent protein tags Various Plasmids Blue/UV , Cyan ...

... tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Conformationally...BRET) CalFlux VNT : An intracellular calcium sensor created by fusing NanoLuc® and Venus fluorophore to ...Luciferase Plasmid Collection You may also like... Fluorescent Proteins Blog: Luciferase 101 Blog: Technologies... in Addgene’s collection are Firefly luciferase (Fluc), Renilla luciferase (Rluc), and NanoLuc® Luciferase...Nano-lanterns , Enhanced Nano-lanterns , and Antares : Fluorescent proteins are fused to Renilla luciferase (Nano-lanterns...-Leu linker to the circularly permuted Venus fluorophore. Learn more about pHlash sensor in our 28 Hot...luciferase with a V5 tag Kevin Janes 83281 pAAV-CAG-FLuc Firefly CAG AAV expression of firefly luciferase...

... Mehl 197576 pAcBac1-Mb-Fluoro-Phe B5 Pyl Fluoro-Phe B5 M. barkeri fluorinated phenylalanine derivatives... Mehl 197577 pAcBac1-Mb-Fluoro-Phe D6 Pyl Fluoro-Phe D6 M. barkeri fluorinated phenylalanine derivatives...5FTrp)RS Methanogenic archaeon 4-Fluoro-L-Tryptophan (4FTrp), 5-Fluoro-L-Tryptophan (5FTrp) Bacterial TAG...7FTrp)RS Methanogenic archaeon 6-Fluoro-L-Tryptophan (6FTrp), 7-Fluoro-L-Tryptophan (7FTrp) Bacterial TAG...TAG Chang Liu 85484 pDule-tfmF A65V S158A tri-fluoromethyl-phenylalanine synthetase M. jannaschii family...Wenshe Liu 137908 PylRS-AS PylRS M. mazei fluorophenylalanine derivatives Bacterial TAG Wenshe Liu 137976...163916 pXPR-SF61 SF5Phe tRNA synthetase para-Pentafluorosulfanyl-Phenylalanine Bacterial TAG Thomas Huber ...

...overexpress various fluorescent markers and tag your proteins of interest with fluorescent proteins in yeast...Teusink Lab Materials Kurt Thorn Lab Materials Fluorescent Protein Plasmids & Resources FP Tagging FP Overexpression...comprehensive collection of yeast codon-optimized fluorescent proteins (FPs) in a variety of plasmids allows...proteins of interest with yeast codon-optimized fluorescent proteins. These vectors are based on the pFA6a-link...Teusink Lab Materials Kurt Thorn Lab Materials Fluorescent Protein Plasmids & Resources External Resources...Goedhart J, Teusink B. In vivo characterisation of fluorescent proteins in budding yeast. Sci Rep. 2019 Feb ... WA, Thorn KS. Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PLoS...

...  APCAlexa Fluor 647   Alexa Fluor 700APC-H7APC-Cy7   Many factors can influence the level of... you'll need to pick a fluorophore for each target and ensure the fluorophores, together, are able to ...reporter's fluorescence. Another way to label proteins is through treating cells with fluorophore-conjugated...dead cells. The combination of antibody-fluorophores, fluorescent reporters, and/or dye stains used in your...outlining a few commonly used fluorophores and their emission spectra. Some fluorophores carry their emission ...and RB545. Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7...respective colors of a few commonly used fluorophores. Each fluorophore has a defined emission maximum. Note...

...fusion protein called CalFlux VNT. CalFlux VNT is composed of Venus fluorescent protein, the Troponin ...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...couple Nluc with a red-shifted fluor. Here, NLuc internally excites the fluor through a process known as ...BRET). The fluor then emits light at a tissue-penetrating wavelength above 600 nm. LumiFluors Schaub, F.X...more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited ... the lab has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed...

...the associated fluorophore or stained with the associated fluorochrome, emits a fluorescent signal that ...the level of fluorescence. A cell expressing high levels of the fluorophore or fluorochrome-stained protein...antibody conjugated fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions...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... to a fluorochrome. In indirect staining a primary antibody binds to a target and a fluorochrome-conjugated... panel of “fluorescence-minus one” controls, samples stained with all of the fluorochromes in the experiment...