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| | | | Phototope®-Star Detection Kit |
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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.
FAQs
- After labeling a probe with the NEBlot Phototope Kit, I am seeing a weak signal. Other than labeling the probe, what else could be the problem?
- After labeling a probe with the NEBlot Phototpe Kit, I am seeing a weak signal; How can I determine if the labeling of the probe is the problem?
- Can I store the NEBlot Phototope Kit at minus 20oC?
- How can I determine if the detection procedure is causing a high or uneven background with the NEBlot Phototope Kit?
- How can I improve the priming reaction in the NEBlot Phototope Kit?
- What are other sources of high or uneven background with the NEBlot Phototope Kit?
- What is the difference between the NEBlot kit and the NEBlot Phototope kit?
- How sensitive are the NEBlot® Phototope® Kits?
- What are the advantages of using a chemiluminescence based detection system versus a radioactive method of detection?
- What is the % of biotinylated dATP in the dNTP mixture provided with the NEBlot® Phototope® Kit?
- What is the rate of biotinylated dATP incorporation in the probed synthesized using the NEBlot® Phototope® Kit?
- Why isn’t the % of biotinylated dATP in the dNTP mxiture higher than 16%?
- Where in the dATP molecule is the biotin attached?
- Are biotinylated dNTPs sold separately?
- How can the biotinylated probe be denatured?
- When I run my probe in a gel, I see a smear. Why?
- What probe size distribution can I expect to obtain by synthesizing a biotinylated probe with the NEBlot Phototope kit?
- Can I produce biotinylated probes by using DNA templates that are shorter than 100 bps?
- Should the unincorporated nucleotides be removed from the biotinylated probe mixture prior to their use for detection?
- Do you recommend the QIAquick PCR purification kit to remove the unincorporated nucleotides?
- Can biotinylated probes be used in gel-shift experiments?
- Is it possible to detect a target RNA or DNA with a biotinylated probe that has been labeled with a single biotin?
Protocols
Protocols for Phototope®-Star Detection Kit
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® Dilution Buffer (25X)
NEBlot® Phototope® Kit
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