Fig. 8.

Recombinant Arabidopsis MDA1 binds ssRNA and dsDNA in vitro. (a) Purification of recombinant MDA1 (rMDA1). rMDA1 was expressed as maltose binding protein (MBP) fusion and purified by amylose affinity chromatography. After TEV protease cleavage, free rMDA1 and MBP were resolved by gel filtration (FPLC). Aliquots of column fractions were analyzed by SDS/PAGE and stained with Coomassie Blue. The elution positions of rMDA1 (c. 100 kDa) and the Conalbumine (77 kDa) and Ovalbumine (44 kDa) molecular weight (MW) markers are indicated below the gel. (b) SDS/PAGE analysis of the purity of the final rMDA1 used for in vitro assays. (c) Gel mobility shift assays showing ssRNA and dsDNA binding of rMDA1. Increasing amounts of rMDA1 (0, 50, 100 and 200 nM) were incubated with synthetic 43‐mer RNA or DNA oligonucleotides of the same random sequence in single‐ or double‐stranded forms and resolved on a native polyacrylamide gel. Free nucleic acids migrate to the bottom of the gel whereas rMDA1‐nucleic acid complexes are shifted up on the gel and are indicated by white diamonds.