Native Gel (nondenaturing conditions):
Gradient gel, 4-20% (native, no SDS)
3.0g Tris (25 mM)
14.4 g glycine (192 mM)
1 liter total -final pH should be 8.8
5x sample buffer
3.1 mL 1 M Tris-Cl (pH 6.8) (312.5 mM)
5 mL glycerol (50%)
0.5 mL 1% bromophenol blue (.05%)
1.4 mL H2O
Typically, 1-5 ug of protein is loaded per well, or up to 30 ug of a complex protein mixture. Fill unused gel lanes with blank sample buffer. Centrifuge sample for 15 min at 10,000 x g prior to preparation for loading in order to remove insoluble material with may interfere.
To load samples, simply mix sample buffer with sample, centrifuge and load. Do not use samples with salt concentration sin excels of 0.1 M as it may cause band distortion.
Run at constant current of about 150 V at 4’C.