 | | | | Phototope®-Star Detection Kit |
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Prices are in US dollars and valid only for US orders.
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Description:
NEB's Phototope Detection for nucleic acids uses biotin associated with the target DNA to provide the handle for the chemiluminescent detection (Figure 1). Biotin can be incorporated directly by enzymatic polymerization of DNA with a biotinylated primer (DNA sequencing) or by polymerization in the presence of biotinylated nucleotide triphosphates (NEBlot® Phototope® Kit, NEB #N7550S).
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, which binds to the streptavidin, is added. This results in a conjugate between the alkaline phosphatase and the DNA on the membrane. Finally, the chemiluminescent reagent (CDP-Star) is added. Alkaline phosphatase catalyzes the removal of a phosphate from the phenylphosphate-substituted 1,2 dioxetane to yield a moderately stable intermediate which then spontaneously decays and emits light. The emitted light is detected by exposing the membrane to X-ray film for 2 to 10 minutes.
A note about membranes:
Phototope Kits require transfer of DNA from a gel to a solid support membrane. The kits were developed using Millipore Immobilon-S membranes; however, other nylon based membranes with a neutral or slightly positive charge can be substituted. Nitrocellulose does not give acceptable results.
Overview of chemiluminescent detection
Advantages:- 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.
Kit Components:
Biotinylated Alkaline Phosphatase
CDP-Star Assay Buffer
CDP-Star® Reagent
Streptavidin
Storage Conditions
Storage Temperature:
4°C
Notes
General notes:- Phototope Kits require transfer of DNA from a gel to a solid support membrane. The kits were developed using Millipore Immobilon-S membranes; however, other nylon based membranes with a neutral or slightly positive charge can be substituted. Nitrocellulose does not give acceptable results.
References
- Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 132, 6-13.
- Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 137, 266-267.
- Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun., 23, 641.
- Bronstein, I., Olesen, C.E.M., Martin, C.S., Schreider, G., Edwards, B., Sparks, A. and Voyta, J.C. (1994) A.H. Campbell, L.J. Kricka and P.E. Stanley (Eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects, pp. 269-272.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), 9.31-9.62.
- 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 (Eds.), Current Protocols in Molecular Biology, pp. 3.19-3.19.8.
Reagents Sold Separately
Biotinylated Alkaline Phosphatase
CDP-Star® Reagent
Streptavidin
Companion Products
CDP-Star® Assay Buffer
NEBlot® Phototope® Kit
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