SDS-page minigel
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实验试剂
1% SDS gel-loading buffer lacking dithiothreitol can be stored at room temperature. Dithiothreitol should then be added, just before the buffer is used, from a 1 M stock
2. Tris-Glycine electrophoresis buffer-1000 ml
A 5X stock can be made by dissolving 15.1 g of Tris base and 94 g of glycine in 900 ml of deionized H2 O. Then, 50ml of a 10% (w/v) stock solution of electrophoresis-grade SDS is added, and the volume is adjusted to 1000 ml withH2 O
1.4M Tris Base, pH'd to 8.9 with HCl
0.5M Tris Base, pH'd to 6.8 with HCl
实验步骤
1. Clean all minigel apparatus with soap and water, then with ethanol.
2. Use a pasteur pipette to seal the bottom of the plates by spreading 1% agarose (made in 4X Tris-glycine buffer) along the bottom of the glass plates so that it goes up the crack by capillary action.
3. Prepare resolving gel (double for two gels):
1.5 ml 4X resolving gel buffer
(mix these in at the same time)
1.5 ml 4X resolving gel buffer
(mix these in at the same time)
4. Quickly add isobutanol to the top of this until the level reaches the top of the plates.
5. Allow resolving gel to polymerize for 20-30 min. TURN ON HEATING BLOCK FOR SAMPLE LOADING LATER.
6. Pour off the isobutanol. Pour water several times into the gel plate space to rinse off all the isobutanol. Dry the watery interplate surface with a piece of Whatmann paper.
1.25 ml 4X solundefined Tris pH 6.8
(mix these in at the same time)
8. Allow the stacking gel to polymerize for 20-30 min.
9. Pour Tris-glycine electrophoresis buffer into the upper and lower chambers. The minigel takes about 200 mL.
10. Before loading the gel flush out the wells with Tris Glycine running buffer
11. When loading the gel, load something (1X loading buffer in blank lanes) in every lane and the dye front will migrate more evenly
13. Run minigel at 15mA. It will take about an hour.
