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We narrowed to 62 results for: Free Use GFP

Showing: 21 - 40 of 62 results

  1. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ... We used the disarmed Agrobacterium tumefaciens strain GV3101(pMP90), which works well for us. Use a well...Fluorescent proteins (FP, e.g. GFP) can be used in many ways. Often a FP is fused to a protein of interest ...can be used to develop stable hairy root lines in tissue culture. This method is commonly used in C. roseus...the commonly used CaMV 35S promoter in Agrobacterium. This can be easily avoided by the use of introns,...gene silencing (VIGS) is a transient technique that uses a virus and the plant's post-transcriptional machinery...Mortensen et al., 2019). Our method might provide some useful guidelines for the development of transient transformation... If you are new to the field, the following are useful tips for your first transient leaf transformation...

  2. Plasmids 101: Protein tags

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    Blog Post
    ...Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection...Affinity Frequently used for western blots, IP, co-IP, IF, flow -cytometry, but rarely used for purification...Fluorescent Protein Should I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein Guide...various mutations used to increase its stability activity, can be readily purified using plasmids found ... proteins can be purified using metal-affinity chromatography, usually using a Ni2+ column. Like other...recognition site. In fact, SUMO cannot be used in eukaryotes because there is already too much SUMO protease...the accompanying cartoon, they have a multitude of uses including (but not limited to) purification, detection...

  3. Better Dyeing Through Chemistry & Small Molecule Fluorophores

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    Blog Post
    ...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...encoded tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 1a). Other strategies...e.g., SNAP-tag, HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme ...be incorporated into a protein structure and then used with the growing toolbox of bioorthogonal chemical...meaning the ligand exhibits low fluorescence when free in solution, but high fluorescence upon binding ...polar groups (Figure 2b), which can preclude their use inside living cells. We recently discovered that ...

  4. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ...simplest and best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, ...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies... If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional...between, you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by ...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...in a freezer box in just about every lab in the world, is not inert. In all seriousness, EGFP/GFP has ...designated mEGFP, mVenus, etc. A word of caution is needed because FPs not derived from GFP, i.e. the ...

  5. Antibodies 101: Immunoprecipitation

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    Blog Post
    ...which can be used for most antibodies compatible with either Protein A or G.  While commonly used, Protein...the antibody used for IP is derived from the same species as the antibody that will be used for detection...such as this, an epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest on the C-...will need to be verified using alternative methods.  Finally, IP can also be used to study proteins that...Immunoprecipitation (IP) uses immobilized antibodies, or immunoglobulins, to isolate a specific protein...protein out of a complex mix. Using this technique, users can look for the presence or absence of a protein...target is present in low amounts. In this method, the free antibody is incubated with the protein mix and antibody-target...

  6. Luciferase Plasmid Collection

    Type
    Collection
    ...emission, substrate use, stability, or other features. AkaLuc : Engineered to use the substrate Akalumine...split-luciferase complementation assay in plants using Gateway cloning. Use with 174051 Alberto Macho 174051 pGWB-cLUC...split-luciferase complementation assay in plants using Gateway cloning. Use with 174050 Alberto Macho 136010 psi-...enhancers using a firefly luciferase reporter. These plasmids improve on the original STARR-seq by using the...Root 105533 pAAV.CMV.Luc.IRES.EGFP.SV40 Firefly CMV AAV expression of firefly luciferase and GFP James Wilson...luciferase have short protein half-lives, making them useful as transcriptional reporters, but are less-than-ideal...promoter/enhancer regions. Blue/white screening can be used for screening of transformants in cloning process...

  7. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...widely used to study gene expression in cells. But these reporters have limited use in vivo because they...genes gvpNJKFGWV marked with Emerald GFP. These plasmids can be used directly in transient transfection ...higher) of BFP and GFP producing cells. After FACS, return the cells to doxycycline-free media and expand...bacteria The first-generation bacterial ARGs (bARGs), used in bacteria, expressed poorly at body temperature...tissue unless destructive ultrasound imaging was used. We set out to develop next-generation bARGs that...expand the cells. Use a subset of the cells to verify the mARG positivity, which can be observed under phase... not require a booster cassette, either, allowing us to reduce the genetic footprint yet again. This ...

  8. Tetracycline Inducible Expression

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    Collection
    ...Iwasato 63704 pRetroX GFP T2A Cre Retroviral vector for dox-inducible expression of GFP T2A Cre recombinase...Eric Kowarz 96930 XLone-GFP Tet-On PiggyBac vector for inducble expression of EGFP Tet-On 3G rtTA TRE3GS...171123 pLVX-TetOne-Puro-GFP Lentiviral Tet-On vector for inducible expression of EGFP Tet-On 3G rtTA TRE3GS...more about the components of tet systems and how to use them, or skip ahead to view highlighted Tet plasmids... Tetracycline On (Tet-On) In 1995, Gossen et al. used random mutagenesis to identify the amino acid residues... interest should only be expressed occasionally, use Tet-On: express an rtTA or TetR and include a tet-responsive...generally active and only turned off occasionally, use Tet-Off: express a tTA and include a tet-responsive...

  9. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore for antibody-conjugated...stains that react with free amines on and inside the cells and are thus typically used for distinguishing ...commonly used fluorophores. Each fluorophore has a defined emission maximum. Note, you can also use the tool...mentioned previously, you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies...you cannot use them together in the same panel. Source: https://www.bdbiosciences.com/en-us/resources/...software (without using the actual FITC fluorophore in our panel) will allow us to detect the eGFP signal. We...the fluorophores used in our panel (Table 2). Source: https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer...

  10. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ...such as GFP (Paix et al., 2014). A further development of this approach reported in 2017 uses the direct...organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us to delete...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...first approach – co-CRISPR from Craig Mello’s lab – used inactivation of the unc-22 gene as their selection...marker (Kim et al., 2014). Meanwhile Andy Fire’s lab used oligo-mediated knock-in of a dominant mutation, ...even more powerful following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with...co-conversion approaches have the advantage of being used in any genetic background, but require variable ...

  11. Quick Guide to Near-Infrared Fluorescent Proteins

    Type
    Blog Post
    ... They can also be used in crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova.... Modern state-of-the-art NIR FPs can be used similar to GFP-like probes by supplying a single gene to... Multiple uses: From multiplexing to multiscaling NIR-shifted spectrum is perfect for use in spectral...35 kDa for miRFPs, and 27 kDa for EGFP) and a possibility to be used as internal tags due to the proximity...near-infrared) (Qian et al., 2019).  NIR FPs are useful in vivo probes, due to low autofluorescence, low...cellular brightness than previously reported widely used dimeric iRFPs (Table 1). In general, the monomeric... endogenous BV.  When imaging NIR FPs, consider using light sources that produce enough light intensities...

  12. Sensing Neuronal Dopamine

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    Blog Post
    ...sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when the receptor ...release.  Using dLight1 sensors in experiments The Tian lab used dLight1 both in brain slices and freely behaving...on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor... for mouse or human receptors, respectively). Using GRABDA sensors in experiments The Li lab used GRABDA...quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive diffusion of DA across the...released DA in mouse brain slices Visualization of DA release dynamics during locomotion in freely moving mice...GRABDA in drosophila, zebrafish, and mouse brain slices and freely moving mice to gather the following types...

  13. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    ...Marker Mouse Strain C-CHIME Knocks out a pair of genes pXPR_219 Violet-excited GFP (vex) ...assessing genes in defined contexts would be quite useful for interrogating these scenarios. X-CHIME We recently...knockout of a single gene pXPR_068 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME ...types (lineage-based) pXPR_070 Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks...of genes sequentially pXPR_220 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME...embryonic lethal, hindering studies post embryogenesis. Using C-CHIME and S-CHIME, we discovered that double knockout..., a suite of four systems which utilizes custom mouse strains and lentiviral expression vectors (Table...

  14. Viral Vectors 101: Calcium Sensors

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    Blog Post
    ...available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free up crowded optical...this blog we will explore some of the most commonly used as well as newly developed calcium sensors. Selecting...calmodulin or troponin C). Their fluorescent readouts are used to monitor calcium concentrations and transients...there waiting for you. When selecting a sensor to use for your studies, you can start by asking ‘What is...changes. The GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters...experiment could be subtle or dramatic. You want to use a calcium sensor that is most sensitive to your expected...available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring only...

  15. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    ...Ready to get started?   Figure 1: Break free from the ball and chain!   Epigenetics nation...Addgene has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre....sorting, and stable gRNA expression can be achieved using lentiviral backbones expressing blasticidin or puromycin...et al. (2016) Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain...clearly testing whether a certain methylation state causes a condition. To understand mechanisms and, eventually...notoriety – as a tool of choice in the field: dCas9 fused to the enzymatic domain of TET1 or TET2 (Morita ... DNA-binding protein with no enzymatic activity cause demethylation?  Dividing cells depend on DNA methyltransferase...

  16. AAV for Neuronal Tracing

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    Collection
    ...Serotype PI 52473 pAAV-synP-FLEX-splitTVA-EGFP-B19G Can be used to complement deletion-mutant rabies virus... 100798 pAAV-syn-FLEX-splitTVA-EGFP-tTA These AAV together can be used to complement deletion-mutant rabies...Ready-to-use AAV available from Addgene's viral service encoding tools for monosynpatic retrograde neuronal...neuronal tracing, to be used with modified rabies virus. Viral...virus-based monosynaptic tracing. These AAV can be used with modified rabies virus to perform retrograde...coated RAVB is released from the starter cell and free to infect neighboring cells via G-protein receptors...viral vector packaging service, but lets us know what viruses would be helpful to the scientific community...

  17. Chromoproteins: Colorful Proteins For Molecular Biology Experiments

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    Blog Post
    ...ambient light. In contrast, the widely used green fluorescent protein (GFP) gives a bright green fluorescence...directly use in bacteria, where most genetic manipulations start. Anthony Forster’s lab used codon optimization...anemone's red color can be used for mimicry. Image: Bernard Spragg. Advantages of using chromoproteins for ...for bacterial synthetic biology Because the common chromoproteins used in the lab come from eukaryotes...fluorescent proteins and luminescence have been widely used in biological research for a long time, chromoproteins...anemone, scientists think that chromoproteins could be used for mimicry and camouflage. An anemone’s red colored... it gives scientists the ability for instrument-free detection. Unlike fluorescence or luminescence, which...

  18. CRISPR 101: Targeting RNA with Cas13a (C2c2)

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    Blog Post
    ...most commonly used CRISPR enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains...starters, Cas13a can be used to bind and cleave target RNAs, although its usefulness in bacterial cells will...LwaCas13a), using a superfolder GFP fusion to stabilize the protein in mammalian cells. LwaCas13a-msfGFP can ...cleave any RNAs after binding a user-defined RNA sequence could be used to detect single molecules of an...exceptional genome engineering tool. It is easy to use, functional in most species, and has many applications...targeting with a single crRNA The Feng Zhang lab used Cas13a from Leptotrichia shahii (LshCas13a) to begin...2016). In the endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate binding ...

  19. The Challenges of Cell Culture

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    Blog Post
    ...cell lines deposited with them and used by different researchers are free of contamination and match their... profiling can be used to ID the cell line. The same test is so good that it is used as a forensic technique...working vials have been used up. This ensures you can retain the maximum stocks and use of the original Cell...for Mammalian Cells Learn about the Dangers of Using GFP for Protein Localization Resources on Addgene.org...the present day Cell culture is a widespread tool used in the fields of oncology, virology, immunology,...specific to their origins. This technique is still used today to test for HeLa contamination. HeLa contamination... world has a unique STR profile) and can also be used as a paternity test. The test is accurate, precise...

  20. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...proteins in these compartments using CRISPR systems should strongly consider using moxFPs over standard FPs ...Generally, FPs evolved in or were engineered for use in cytoplasmic environments. However, approximately...green, cyan, yellow, and blue variants that can be used in various combinations. For example, with standard...herpesvirus.       References 1. Snapp, Erik. "Design and use of fluorescent fusion proteins in cell biology."Current...: PMC2784028. 3. Munro, Sean, and H. R. Pelham. "Use of peptide tagging to detect proteins expressed from...Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when targeted...Suzuki, Takahisa, et al. "Development of cysteine-free fluorescent proteins for the oxidative environment...
Showing: 21 - 40 of 62 results