The flow-through retained from the RNA binding column now contains just the protein fraction. Purification of this is based on a straightforward precipitation protocol, the flow-through first being mixed with a supplied precipitation reagent and then spun at 16,000 g to pellet the precipitated proteins. The pellet is washed with pure water before resuspension. The triplePrep kit protocol recommends resuspending in a 2D-DIGE buffer for optimal results, although 7M urea can also be used if required.
Yields are expected to vary depending on the sample type but the key question for researchers with precious samples, who need to maximize yield is how the three-in-one approach compares to traditional methods. The triplePrep kit was compared to established kits and protocols for the purification of DNA, RNA, and protein as individual analytes, with the finding that not only does triplePrep equal these methods in yield but it exceeds them in many cases.
Purity and Reliability
The ease of purification, and the yields expected from different sample and tissue types varies. Those that are more metabolically active contain higher amounts of RNA and protein, while those that are more fibrous in nature are more difficult to disrupt effectively and so tend to have lower overall yields for all three analytes. Any purification method should maintain the overall quality and purity of the isolated analyte despite these differences in the starting sample. Analysis of results from a wide range of starting materials commonly used in research applications shows that the triplePrep kit returns not just high yields but also consistent purity throughout (Table).
The final test of the purified analytes is how they perform in downstream analysis. During testing, the isolated DNA, RNA, and denatured proteins were found to be entirely compatible with end-point PCR, sequencing, real-time PCR, microarray analysis, SDS-PAGE, Western blotting, 2D-DIGE, and LCMS. DNA, RNA, and protein purified using the triplePrep kit were compared with that purified using single-analyte purification methods. In all cases, the data collected from triplePrep samples gave results directly comparable to, or better than, those from traditional approaches. For example, protein analysis was conducted by 2-D DIGE analysis against the recommended 2-D DIGE reference sample (Figure 2).
Isolating and purifying DNA, RNA, and protein from a single sample provides real advantages, not just in the time required to perform the purification itself, but also in reducing the amount of sample that must be used in these processes. This presents a real advantage in situations where samples are in short supply, difficult to replace, or simply unique. For samples whose heterogeneity can lead to questions on how readily RNA, DNA, and protein results can be correlated from separate purification processes, the outcome of using the triplePrep kit is a greater certainty in comparing results and analysis. All of these are possible without any reduction in the yield, quality and performance of the purified analytes.