The Alkphos Direct Labeling And Detection System from GE Healthcare is a detection method for labeling probes to be used for Northern or Southern blots. The main advantage of this kit is that does not require the use of radioactive labels but instead directly labels RNA or DNA probes with a thermostable alkaline phosphatase (AP) enzyme by crosslinking the enzyme to the nucleic acid. The AP enzyme can then be detected using a chemiluminescent substrate that comes with the kit. Two different detection reagents are available - the CDP-Star reagent for high to medium sensitivity or the ECF detection substrate for fluorescence. There is no difference in the amount of time it takes to detect a probe between the two reagents - with either one, it takes just 30 minutes from completion of hybridization to application of the detection reagent. Another advantage of the Alkphos Direct kit is that when using the CDP-Star chemiluminescent substrate, dioxetane, the light output reaches a maximum at 4 hours but then lasts for several days. This allows for multiple, long exposures where other chemiluminescent systems, utilizing HRP, only last about 6-8 hours.
We have used this kit in our lab for both northern and Southern analysis. In our hands, the Southern analysis has worked beautifully but we have had problems using this system for Northern blots. With the Southern blots, the kit detected my transcripts wonderfully, although I was not probing a very complex sample (I used a DNA labeled probe to identify cloned DNA that had been run out on a gel). We use the Southern to identify tumor antigens (from multiple samples) in our lab in order to save money on sequencing 30 or more clones. After using the kit a few times, the labeling, hybridization and washing steps went quickly and proved to be easier than dealing with radioactive probes, blots and washes. You just can’t beat the cost of this kit versus the hazard of working with radiolabeled probes not to mention the cost, time and effort it takes to deal with radioactivity. Also, the AP-labeled probes can be stored and used for 6 months. Our experience with using this kit for northern blots wasn’t as good. The Alkphos direct kit was not nearly as sensitive (in our hands) as a [32]P labeled probe. If you are detecting an abundant transcript and have plenty of RNA to play around with the hybridization conditions, this kit may be for you. However, if your RNA is scarce along with the transcript, stick with radiolabeled probes.
In summary, while this kit worked well for us as a substitute for radiolabeled probes in Southern blot analysis it did not work for us in our northern blot analysis. Although, because our RNA samples are precious, we did not try to optimize conditions, which may have helped. But for Southern blots, this kit can save time, money and it allows you to avoid having to work with radioactivity.
Alicia Racelis
Medical Genetics Graduate Student
University of Alabama at Birmingham