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. 2020 Jul 1;11(7):734. doi: 10.3390/genes11070734

Figure 4.

Figure 4

Protein analyses of the poly-histidine (His)-small ubiquitin-like modifier (SUMO)-tagged Dicer-related helicase (DRH)-1 and DRH-1 isoform b eluted from anion-exchange column chromatography. The affinity-purified His-SUMO-tagged DRH-1 (a) and DRH-1 isoform b (b) were further purified after dialysis using anion-exchange column chromatography. The proteins in 4.5 μL aliquots of the indicated fractions, the dialyzed proteins loaded into the column (D), and the pass-through fraction (Pt) were analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue (CBB) staining (left) and western blotting with peroxidase-labeled anti-His-tag antibody (right). The pooled peak fractions (numbers 24–30) of both His-SUMO-tagged DRH-1 and DRH-1 isoform b were concentrated using ultrafiltration, and then analyzed as already described (CBB staining, left and western blotting, right in panel c). The affinity-purified and concentrated His-tagged DRH-3 was also analyzed using 12% SDS-PAGE followed by CBB staining and western blotting in panel c. WIDE-VIEW Pre-stained Protein Size Marker III (Wako Pure Chemicals), MagicMark XP Western Protein Standard (Thermo Fisher Scientific), and Precision Plus Protein Standards (Bio-Rad) were used as molecular weight markers (M) for CBB staining and Western blotting in panels (a, b and c), respectively. Arrowheads indicate the predicted molecular weights of the tagged proteins: His-SUMO-tagged DRH-1, 131.3 kDa, His-SUMO-tagged DRH-1 isoform b, 101.1 kDa, and His-tagged DRH-3, 132 kDa.