Yu:SDS PAGE

SDS-PAGE

Casting Gel

Casting 2 SDS-PAGE Gels with ProtoGel

1. Assemble the gel-casting apparatus	Assemble casting apparatus
2. In a 15mL tube, prepare resolving gel for the desired percentage:	Mix Resolving Gel
Resolving Gel 12% 10% 8% 7% ProtoGel 4mL 3.3mL 2.7mL 2.3mL Millipore H2O 2.7mL 3.3mL 4mL 4.4mL 1.5M Tris pH 8.8 2.4mL 10% SDS 100μL TEMED 10μL
3. Add 50μL 10% APS and pour resolving gels immediately. APS catalyzes the polymerization of the gel	Add 50μL 10% APS and pour gel
4. Pour resolving gels to approximately 2-3 cm below the edge of the gel cassette. The space above is required for stacking gel
5. Add a thin, .5 cm layer of isopropanol to the gel cassettes immediately after pouring the resolving gel. This ensures that the top edge of the resolving gel is level, free of bubbles, and polymerizes evenly	Add isopropanol
6. While the resolving gel is polymerizing, prepare stacking gel in a 15mL tube:	Mix Stacking Gel
Stacking Gel (5%) ProtoGel 650μL Millipore H2O 3.2mL 1.5M Tris pH 6.8 475μL 10% SDS 37.5μL TEMED 5μL
7. Wait for the resolving gel to fully polymerize. When the leftover stacking gel in the tube has solidified and tilting the casting apparatus from left to right does not cause the gel to shift, polymerization is complete
8. Gently wash isopropanol out of the gel cassette with millipore H2O and dry with a kimwipe	Remove isopropanol after polymerization
9. Add 25μL 10% APS to the stacking gel in the tube. Pour stacking gels immediately, as APS catalyzes polymerization	Add 25μL 10% APS and pour
10. Add a comb containing the desired number of wells immedietely. Ensure that no bubbles or air pockets are trapped under the comb	Add gel comb
11. Allow stacking gel to fully polymerize before using. Gels can be stored wrapped in damp paper towels in a 4°C refrigerator for a week or more