The Strip-EZ technology from Ambion involves synthesizing DNA and RNA probes (which they call StripAble probes) that can be easily removed from nucleic acid blots. Ambion currently offers the Strip-EZ technology in four different kits: a random-primed DNA probe synthesis kit (Strip-EZ™ DNA Kit), an in vitro transcription for RNA probes kit (Strip-EZ™ RNA Kit), a PCR probe synthesis kit (Strip-EZ™ PCR Kit) and a cDNA probe synthesis kit (Strip-EZ™ RT Kit). These kits provide all of the reagents necessary to produce StripAble probes for use in various nucleic acid blots (e.g. Southern blots, Northern blots, dot-blots, etc.). StripAble probes are synthesized in the same way as traditional probes except that a modified dCTP is incorporated in the DNA probes and a modified CTP is incorporated in RNA probes. StripAble probes are stable under the conditions used for blot hybridization, washing and signal detection. These kits also supply reagents to cleave StripAble probes hybridized to target molecules on a blot after hybridization and detection. Degradation is specific to the probe because cleavage occurs at the modified nucleotides (i.e. modified dCTP or CTP) that are incorporated during probe synthesis. StripAble probes are removed in mild wash conditions, so blots can be used again to detect other targets.
I often use Strip-EZ kits to synthesize StripAble probes (both DNA and RNA probes) with 32P-labeled nucleotides. The quality, stability and specificity of the 32P-labeled StripAble probes are equivalent to those of 32P-labeled probes synthesized by traditional methods. The presence of the modified nucleotides in StripAble probes does not appear to affect its hybridization to the blot. In my hands, these probes work well in various downstream applications, including Northern/Southern Blotting, microarrays and library screening. The stripping procedure provided with the kit is simple and following it I could successfully remove probes from blots under very mild wash conditions (68C for 10min). Importantly, these probes appear to work well for various membranes made by different manufacturers. I always expose the membranes to film or a phosphoimager after the stripping procedure to ensure that the background is low or non-existant and I have not had a problem.
The synthesis and purification procedures used for 32P-labled StripAble probes are the same as those used for other 32P-lableing kits except for the incorporation of the modified nucleotides. According to Ambion, these kits can also be used for non-isotopic labeling, but I have not tried this yet. One thing to keep in mind is that it is important that the membrane is kept wet after hybridization and during the stripping process. In addition, I generally make all of the buffers fresh.
Current methods to strip a probe from a blot include harsh treatments, such as boiling in an SDS solution. These protocols often cause irreversible damage to the blot that effectively reduce its re-usability. The Strip-EZ kits provide a good solution to this problem. This technology allows you to re-probe blots repeatedly, allowing you to get more information from your experiments.
Yng-Ju Hsieh
Postdoctoral Fellow
Rockefeller University