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  • Phototope®-Star Detection Kit 

    Discontinued Date

    04/16/2013
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    Categories:
    Discontinued Products

    Description

    Phototope® Detection is based on chemiluminescence, an enzyme-catalyzed reaction which emits a blue light. This detection method is replacing radioactive detection due to sensitivity, convenience, safety and cost.
    In the Phototope Detection Kits for nucleic acids, biotin associated with the target DNA provides the handle for the chemiluminescent detection. Biotinylated DNA is detected on a membrane support by first exposing the membrane to streptavidin, which binds to the biotinylated DNA. Next, biotinylated alkaline phosphatase complex is added, which binds to the streptavidin, resulting in the creation of a conjugate between the alkaline phosphatase and the DNA on the membrane. In the final step, the CDP-Star™ reagent is added. Alkaline phosphatase catalyzes the removal of the phosphate from the CDP-Star® (phenylphosphate substituted 1,2 dioxetane) to yield a moderately stable intermediate which then spontaneously decays, emitting light at 461 nm. The emitted light is detected by exposing the membrane to X-ray film for 1 to 10 minutes.

    The biotin handle can be associated with your target DNA either directly (covalently) or by hybridization. Biotin can be incorporated directly into DNA by using enzymatic polymerization of DNA with a biotinylated primer (for DNA sequencing) or by polymerization in the presence of biotinylated nucleotide triphosphates (as with the NEBlot® Phototope® System).

    Overview of chemiluminescent detection

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Streptavidin -201 mg/ml
    Biotinylated Alkaline Phosphatase40.5 mg/ml
    CDP-Star® Dilution Buffer (25X)425X

    Advantages and Features

    Features

    • Stability: Biotinylated probes and reagents are stable for extended periods unlike 32P-labeled probes which are only stable for a few days.
    • Speed: Only 40 minutes is required for the entire detection procedure. Exposure times of 1-10 minutes.
    • Multiple Exposures: Light is emitted at a constant rate for days, so you can perform multiple exposures to optimize signal intensity. Re-exposure at a future date is achieved by simply adding more chemiluminescent reagent.

    Properties and Usage

    Storage Temperature

    4°C

    Notes

    1. Phototope Kits require transfer of DNA from a gel to asolid support membrane. The kits were developed using Millipore Immobilon-Smembranes; however, other nylon based membranes with a neutral or slightlypositive charge can be substituted. Nitrocellulose does not give acceptableresults.

    References

    1. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem.. 132, 6-13.
    2. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem.. 137, 266-267.
    3. Denhardt, D.T. (1966). Biochem. Biophys. Res. Commun.. 23, 641.
    4. Bronstein, I., Olesen, C.E.M., Martin, C.S., Schreider, G., Edwards, B., Sparks, A. and Voyta, J.C. A.H. Campbell, L.J. Kricka and P.E. Stanley(Ed.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. 269-272. Chichester, England: John Wiley and Sons, Inc..
    5. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd Ed.). 9.31-9.62.
    6. Perry-O'Keefe, H. and Kissinger, C.M. (1989). F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl(Ed.), Current Protocols in Molecular Biology. 3.19-3.19.8. New York: Wiley-Interscience.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].
    1. Detection using the Phototope-Star Detection Kit
    The component in the detection kit most sensitive to freezing is the biotinylated alkaline phosphatase (N7022S). It should survive one freezing but certainly not repeated freezings. A quick detection test by serial dot blotting biotinylated 2-log ladder (N7554S, diluted in 0.1 N NaOH) on a membrane is recommended.