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. 2015 Jan 23;10(1):e0116736. doi: 10.1371/journal.pone.0116736

Figure 1. Purification of native DCLs from wheat germ extracts.

Figure 1

a) Gel filtration analysis of crude wheat germ extract using Sephacryl S300. Fractions were collected and incubated with radiolabelled RNA substrates. The processed RNAs were precipitated with isopropanol, washed with 80% ethanol and dissolved in 10ul of loading dye (100% deionized formamide and 0.1% bromophenol blue) before separating on a 15% polyacrylamide gel. OD of the fractions was calculated separately and merged as green line with the gel picture. b) Determination of the sizes of DCL complexes. Three known marker proteins were used to draw a standard curve and sizes of DCL complexes was inferred using this standard curve. c) Ammonium sulfate precipitation to separate 24 and 21 nt generating activities. Please see methods for details. Processed RNAs were detected as described above.