RNaseAlert Lab Test

If you fear your protein has some nuclease activity, the easiest way to test is using the RNaseAlert Lab Test kit (Cat# AM1964) from Ambion (now Applied Biosystems, now Life Technologies). If you perform a +/- protein selection during a round and find the sample without protein amplifies much sooner than with protein it indicates the protein is somehow interfering with amplification, i.e. chewing up your target somehow. This can happen if the target prep is not very pure or the target is similar size to RNase (14, 37, 125 kDa).

Using the protocol, you can compare the fluorescence of your target sample with a sample incubated with RNase (+ control) or water (- control). Each sample should be around 50ul with 5ul 10X buffer. To read the sample, you will need to use a microplate reader. Since you need two samples in addition to your number of proteins, its good to include as many protein samples as you can find (i.e. ask your lab mates to test their targets too. It's good data).

We have the BioTek Synergy HT for this. Set up a kinetic plate read and take measurements every 5 minutes for 1 hour under conditions similar to your selection. I like to use the Corning 96 well half area flat bottom plates for these assays (Cat # 3686). The water should show no fluorescent activity after 30 minutes whereas the RNase sample should show full fluorescence after 5-10 minutes. You can compare your sample when the difference is greatest between the +/- controls or after the length of time you typically leave your binding reaction.

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