Ambion’s BrightStar BioDetect Kit is designed for nonisotopic detection of biotinylated RNA and DNA probes on positively charged nylon membranes. Ambion claims to reduce background noise and increase sensitivity with this kit, as compared to other nonisotopic chemiluminescent detection methods. The kit comes complete with all of the reagents required for detection following binding of the biotinylated nucleic acids (probe is hybridized to membrane-specific target). This kit can be used for blot hybridizations as well as ribonuclease protection assays.
As with any protocol, this kit requires all containers to be cleaned with RNaseZap and rinsed with RNase free water. The post-hybridization washes should be completed before starting the BioDetect procedure. The procedure requires 2 washes in 1X wash buffer and 2 washes in blocking buffer at room temperature with constant agitation. After a 30 minute incubation in the blocking buffer, the membrane is incubated in diluted Streptavidin-Alkaline Phosphatase and subsequently washed. The membrane is incubated for 5 minutes in CDP-Star and then may be exposed to radiographic film within 2-48 hours. All of the reagents come either ready to use or require a simple dilution with de-ionized water.
I used this kit to detect a biotinylated probe hybridized to a Northern blot. I used Ambion’s BrightStar Psoralen-Biotin Nonisotopic Labeling Kit to label the DNA probe and Ambion’s NorthernMax kit to run the gel and blot. I also used the recommended Ambion Bright-Star-Plus positively charged nylon membrane. I have previously done Northern blots the laborious way (making all the reagents, cutting the membranes and ordering chemicals) and the detection process took multiple attempts to reduce background and increase sensitivity. When I performed the Northern blot using this system, the result was a perfect blot on the first try. There was minimal background and high sensitivity. The kit itself is not expensive relative to the reduced bench time and increased ease. I found this kit to be very easy to use and a necessity for laboratory technicians that only run an occasional blot and do not have the time to troubleshoot and tweak the protocol. The manual specifically stated all the required times and reagent amounts and had a thorough troubleshooting section if required. This kit reduces prep time by days as it comes with fully prepared reagents that can be quickly diluted. It also reduces the number of containers that need to be prepped and cleaned. I highly recommend this kit along with the entire Ambion NorthernMax system for any laboratory.
Katrina Williams
Lab Specialist, Sr
McGuire VAMC
Richmond, VA