[molecular biology work] zymography1

[Seung Jae Jeong]

Zymography for KRS (MMP-9, -2)

2X10⁵ of Raw 264.7 per well (12 well dish) were seeded in DMEM containing 10% FBS at 37 degree in a 5% CO2 atmosphere.

Before treatment of KRS, cells were kept in serum free DMEM for 3hrs and KRS was treated for 24hrs 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 8% 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.

 

MMP-1 assay : 2mg/ml of casein in SDS PAGE

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