2.3. Gel retardation assay
The formation of siRNA/peptide complexes was confirmed by gel
retardation assay. Total 10 μL complexes of 10 pmol siRNA and each
peptide were self-assembled with a range of N/P ratios (1:1, 5:1, 10:1,
20:1, 30:1, 40:1, 50:1, and 100:1) as mentioned above. After adding 6X
loading dye, total 12 μL complexes were loaded into 2% agarose gel
(w/v) prepared in 1X TAE buffer (40 mM tris, 20 mM acetic acid, 1 mM
EDTA, pH=8.6) with 10,000X TopRed Nucleic Acid Gel Stain for
visualization. Gel running was performed at 25 VA for 30 minutes using
the Mupid-2plus electrophoresis system (Optima Inc., Tokyo, Japan). The
electrophoretic mobility shift of complexes was taken pictures by
ChemiDoc™ XRS+ System (Bio-Rad, CA, USA).