Yu:Western

Western Blotting

SDS PAGE

1.Cast SDS-PAGE gel and assemble the gel apparatus.

2. Add 10μL of desired sample and 5μL 3x Gel loading buffer to an eppendorf tube and boil for 5 minutes. Do this in separate tubes for each sample to be analyzed. After boiling, samples may be kept at room temperature until loaded

3. Load 12μL to 15μL of sample into each lane. Load protein prestain marker, as well as BenchMark, if desired. Load any empty lanes with 1x Gel loading buffer

4. Run gel until the loading buffer is just running off the bottom of the gel

Stripping

Stripping Buffer

Solution	Volume
1.5M Tris pH 6.8	2.08 mL
β-Mercaptoethanol	0.5 mL
10% SDS	10 mL
milliQ H2O	37.42 mL
Final Volume is 50 mL

Procedure

1. Wash Membrane for 15-20 min in 1x PBST at room temperature to wash away developing solution
2. Incubate membrane in 50 mL of stripping buffer on a shaking platform for 30 min at 50°C
3. Wash Membrane for 15-20 min in 1x PBST at room temperature
4. Block and reprobe membrane with desired antibody