How RNaseAlert Lab Test works
Rapid RNase detection is achieved using a cleavable fluorescent-labeled RNase substrate developed as a collaborative effort by Ambion and Integrated DNA Technologies, Inc. (patent pending). The RNaseAlert Lab Test assay is simple and straightforward; just pipet 5 μL of the 10X RNaseAlert Lab Test Buffer into one of the tubes containing lyophilized Fluorescent Substrate. Then, add up to 45 μL of the solution to be tested, and incubate for 30 min to 1 hr at 37°C. The Fluorescent Substrate is a modified RNA oligonucleotide that emits a green fluorescence if it is cleaved by RNase, the fluorescence can be visually detected by short-wave UV illumination or measured in a fluorometer. Solutions with RNase contamination will produce a green glow in the assay, whereas solutions without RNase activity will not fluoresce. The amount of RNase activity will be directly proportional to the rate of fluorescence increase. Quantitative measurements can be obtained from a fluorometer.
A positive (+) control, negative (-) control, glycogen and five different fluorescent proteins (mTagBFP, mKate, mTagGFP, mCherry, EGFP) were used in this test. The samples were placed in a Corning 96 well half area flat bottom plate and examined for fluorescent activity in the BioTek Synergy HT. The kinetic plate was programmed to take measurements every five minutes for one hour. The results, in graphical form, were as follows:
NOTE: mTagGFP and EGFP were disregarded in some of these data analyses due to its own excitation at the same wavelength and thus background fluorescence.
As can be seen, the only protein with any significant fluorescence and thus RNase activity was mTagBFP(the protein I am doing my selection with).
Over a 30 minute period, one can see that the extent of the RNase activity of mTagBFP is approximately 10-11% of the positive control. In other words, 10% of my binders are being chewed up.
Interestingly, EGFP and mTagGFP did not react in the same manner. One would expect photobleaching, as seen with EGFP, since the protein was constantly fluorescing during the test. However, mTagGFP did not exhibit these results.
1 comment:
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RNase A is an RNase that is commonly used in research. RNase A (e.g., bovine pancreatic ribonuclease A: PDB 2AAS) is one of the hardiest enzymes in common laboratory usage; one method of isolating it is to boil a crude cellular extract until all enzymes other than RNase A are denatured. It is specific for single-stranded RNAs. It cleaves the 3'-end of unpaired C and U residues, ultimately forming a 3'-phosphorylated product via a 2',3'-cyclic monophosphate intermediate.
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
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