[박상규] Zymography

[Anonymous]

Zymography

1X10⁵ of BAEC per well were seeded in DMEM containing 20% FBS at 37 degree in a 5% CO2 atmosphere. Before treatment of p43, cells were kept in DMEM containing 2% FBS for 2hrs and p43 was treated for 24 hrs as indicated concentration. 20ul of medium was mixed with 5X FOD buffer(4% SDS, 20% glycerol, 0.01% bromo phenol blue, 0.125M Tris-HCl, pH 6.8) and run to the 10% SDS-PAGE containing 1mg/ml of gelatin. After running, the gel was washed with 2.5% Triton X-100 two times for each 20 min and washed again with D.W two times for each 20 min. After washing, the gel was incubated with reaction buffer(50mM Tris-HCl, pH 7.5, 10mM CaCl2, 150mM NaCl, 1uM ZnCl2, 1% Triton X-100, 0.002% sodium azide) for 24 hrs at 37 degree. The gel was washed with D.W and stained with coomassie blue R250 and destained with 35% methanol.

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