Gel polymerization and gel running buffers were of the same composition (50 mM HEPES and 20 mM Tris, pH 7.0). Native 6% PAGE gels were prepared by first making a gel solution containing 6% acrylamide (29:1) (Fisher Scientific) (16 (link)). This solution was then degassed under vacuum while stirring. 12 ml of degassed solution (enough for two gels) was polymerized by mixing with 200 μl of 10% (w/v) ammonium persulfate (Biorad) dissolved in water and 10 μl of TEMED (Acros).
A set of 10 μl reactions containing varying amounts of Cre variants (1–1 000 nM) and 10 nM 5′-Cy5-labeled (bottom strand) loxP DNA (IDT), were prepared in binding buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 2 mM DTT, pH 7.0) along with 50 μg/ml salmon sperm DNA (Invitrogen) and 100 μg/ml BSA (Thermo Scientific) (16 (link),44 (link)). Binding reactions were equilibrated at room temperature for 30 min. 1 μl of 10× EMSA gel loading dye (60% glycerol, 40% binding buffer, 0.1% bromophenol blue) was added to each reaction and then loaded onto a pre-run (120 V, 30 min) 6% native PAGE gel using gel running buffer. Samples were electrophoresed at 80 V for 75 min and imaged on an Amersham Typhoon Gel imager.
A set of 10 μl reactions containing varying amounts of Cre variants (1–1 000 nM) and 10 nM 5′-Cy5-labeled (bottom strand) loxP DNA (IDT), were prepared in binding buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 2 mM DTT, pH 7.0) along with 50 μg/ml salmon sperm DNA (Invitrogen) and 100 μg/ml BSA (Thermo Scientific) (16 (link),44 (link)). Binding reactions were equilibrated at room temperature for 30 min. 1 μl of 10× EMSA gel loading dye (60% glycerol, 40% binding buffer, 0.1% bromophenol blue) was added to each reaction and then loaded onto a pre-run (120 V, 30 min) 6% native PAGE gel using gel running buffer. Samples were electrophoresed at 80 V for 75 min and imaged on an Amersham Typhoon Gel imager.